Vaccine comprising beta-herpesvirus

ABSTRACT

The present invention relates to a beta-herpesvirus, preferably a recombinant beta-herpesvirus, wherein the beta-herpesvirus comprises at least one heterologous nucleic acid, wherein the at least one heterologous nucleic acid comprises a gene encoding a cellular ligand.

The present invention is related to a beta-herpesvirus comprising atleast one heterologous nucleic acid, the beta-herpesvirus for use in amethod for the treatment and/or prevention of a disease such as HIVinfection, influenza virus infection, HPV infection, Helicobacter pyloriinfection, Mycobacterium tuberculosis infection and/or Plasmodiumfalciparum infection, the use of the beta-herpesvirus, a nucleic acidcoding for the beta-herpesvirus, a vector comprising the nucleic acid, ahost cell comprising the nucleic acid and a pharmaceutical compositioncomprising the beta-herpesvirus, the nucleic acid and/or the and apharmaceutically acceptable carrier.

Human cytomegalovirus, also referred to herein as HCMV or human CMV, isan important human pathogen causing morbidity and mortality incongenitally infected and immunosuppressed individuals.Cytomegaloviruses, also referred to herein as CMV(s), are highly adaptedto their mammalian hosts and are host-species specific in theirreplication which precludes the study of HCMV in animal models. Researchon murine cytomegalovirus, also referred to herein as MCMV. Mouse CMV ormurine CMV is the most advanced model in regard to the principles thatgovern the immune surveillance of CMVs. After primary infection the hostimmune response effectively terminates virus replication; however,clearance of the viral genome is not achieved and CMV is able toestablish lifelong latency with periodic reactivation and shedding ofvirus (Pass, R. F. et al. 2001, in Fields Virology. D. M. Knipe and P.M. Howley, editors. Philadelphia: Lippincott Williams and Wilkins.2675-2706, Stratton K R et al., in Vaccines for the 21st Century: A Toolfor Decision Making: Report of the Committee to Study Priorities forVaccine Development, Washington, D.C., USA: Institute of Medicine;1999).

While HCMV infection is readily controlled by the immune competent host,the virus displays its pathogenic potential when host immunity isimpaired. HCMV infection is the most common viral congenital infectionwhich may result in live-long neurological sequelae, including braindamage, sensorineural hearing loss and mental retardation (Boppana, S.B. et al., 1997, Pediatrics 99:409-414; Boppana, S. B. et al., 1992,Pediatr Infect Dis J 11:93-99; Hamprecht, K. et al., 2001, Lancet357:513-518; Whitley, R. J, 2004, Adv Exp Med Biol 549:155-160). Solidorgan transplant recipients and hematopoietic stem cell transplantrecipients are the second group of patients at risk for severe CMVinfections (Mwintshi, K., and Brennan, D. C., 2007, Expert Rev AntiInfect Ther 5:295-304; Ljungman, P., 2008, Bone Marrow Transplant 42Suppl 1:S70-S72; Streblow, D. N. et al., 2007, Curr Opin Immunol19:577-582). In HIV-infected patients CMV continues to be the mostfrequent viral opportunistic pathogen although severe infections havebecome less common following the introduction of highly activeantiretroviral therapy (Steininger, C. et al., 2006, J Clin Virol37:1-9). Due to this immense public health importance, the developmentof a HCMV vaccine has been ranked as a top priority for the 21st centuryby the US Institute of Medicine (Arvin, A. M. et al., 2004, Clin InfectDis 39:233-239).

Both innate and adaptive immune responses are important for the controlof CMV infection (Krmpotic, A. et al., 2003 Microbes Infect 5:1263-1277;Koszinowski, U. H. et al., 1991, Curr Opin Immunol 3:471-475; Reusser,P. et al., 1991, Blood 78:1373-1380; Einsele, H., 2002, Cytotherapy4:435-436; Peggs, K. S. et al., 2003, Lancet 362:1375-1377). Innateimmunity, in particular NK cells, plays a key role in limiting CMVinfection at an early stage and in priming of the adoptive immuneresponse (Robbins, S. H. et al., 2007, PLoS Pathog 3:e123; Rolle, A.,and Olweus, J., 2009, APMIS 117:413-426).

NK cells play a crucial role in fighting many pathogens. As part of theinnate immune system they represent the first line of a host defense.Their activation is a result of signal balance from their inhibitory andactivating receptors. Healthy and untransformed cells express MHC classI molecules which bind to NK cell inhibitory receptors providinginhibition of their activity. When MHC class I molecules are lackingfrom the cell surface, or cells express ligands for activating receptorson NK cells, NK cells are activated to kill infected or transformedcell.

One very potent activating receptor, whose engagement can overrideinhibitory signals, is NKG2D. In addition to NK cells, NKG2D is alsoexpressed on γδ T cells and activated CD8⁺ T cells, where it hasco-stimulatory function. Ligands for the NKG2D receptor in mice are themembers of the RAE-1 family of proteins (RAE-1α, β, γ, δ, ε) as well asMULT-1 and H60 proteins. NKG2D plays a major role in fighting MCMV. Thebest evidence for the importance of NKG2D is given by the fact that MCMVevolved numerous mechanisms against NKG2D (Lisnic, V. J. et al., 2010,Curr Opin Microbiol 13(4):530-9).

CD8⁺ T cells are the principal effectors required for resolution ofproductive infection and establishment of latency (Reddehase, M. J.,2002, Nat Rev Immunol 2:831-844). Although CD8⁺ T cells play a dominantrole, CD4⁺ T cells and NK cells contribute to the maintenance of latentCMV infection (Polic, B. et al., 1998, J Exp Med 188:1047-1054).

Antiviral antibodies, although not essential for the control of primaryCMV infection and the establishment of latency, play a critical role inlimiting the dissemination of recurrent virus (Jonjic, S. et al., 1994,J Exp Med 179:1713-1717). Antibodies can modify the disease associatedwith HCMV infection in transplant recipients as well as congenital CMVinfection in humans and experimental animal models (Nigro, G. et al.,2005, N Engl J Med 353:1350-1362; Nigro, G. et al., 2008, Prenat Diagn28:512-517; Bratcher, D. F. et al., 1995, J Infect Dis 172:944-950;Cekinovic, D. et al., 2008, J Virol 82:12172-12180; Chatterjee, A. etal., 2001, J Infect Dis 183:1547-1553; Snydman, D. R. et al., 1987, NEngl J Med 317:1049-1054).

Consequently, a CMV vaccine should ideally aim to elicit an effectivecellular and humoral immune response and at the same time the CMVvaccine should be safe for the vaccinated patient.

A number of subunit vaccine strategies and live, attenuated CMV vaccineshave been developed (Schleiss, M. R., 2008, Curr Top Microbiol Immunol325:361-382; Zhong, J., and Khanna, R., 2007, Expert Rev Anti InfectTher 5:449-459; Gonczol, E., and Plotkin, S., 2001, Expert Opin BiolTher 1:401-412; Griffiths, P., 2009, Rev Med Virol 19:117-119; Mohr, C.A. et al., 2010, J Virol 84(15):7730-42). Recently, a phase 2 clinicaltrial was described that suggested a protective capacity againstmaternal infection by use of recombinant monovalent glycoprotein B HCMVvaccine (Pass, R. F. et al., 2009, N Engl J Med 360:1191-1199).Glycoprotein B is also referred to herein as gB. While subunit vaccinesinduce an immune response to selected viral proteins, the advantage oflive vaccines is that they elicit an immune response that mimics naturalimmunity and provides a broader protection. Their use, however, carriesthe risk of CMV disease caused by the vaccine strain or reactivation inthe immunocompromised state, unless residual immunity would efficientlycontrol the vaccine virus.

One approach to generate such an immunogenic, yet safe live vaccine isby deleting viral genes that subvert the host immune response(Cicin-Sain, L. et al., 2007, J Virol 81:13825-13834; Crumpler, M. M. etal., 2009, Vaccine 27:4209-4218) or essential genes resulting inspread-deficient virus (Mohr, C. A. et al. 2010, supra).

Nevertheless, until today there is no approved HCMV vaccine availableand such HCMV vaccine has been recently ranked as a top priority for the21st century by the US Institute of Medicine (Arvin, A. M. et al., 2004,supra; Stratton, K. R. et al., 1999, supra).

Thus a problem underlying the present invention is to provide aneffective HCMV vaccine and a beta-herpesvirus contained in such vaccine,respectively.

It is also a problem underlying the present invention to provide abeta-herpesvirus being an effective vaccine against infection with apathogen in addition to or other than HCMV, wherein the pathogen isselected from the group comprising viruses other than HCMV, bacteria andparasites.

It is a still further problem underlying the present invention toprovide a nucleic acid coding for a beta-herpesvirus, wherein thebeta-herpesvirus is suitable for use as an effective HCMV vaccine.

These and other problems underlying the instant invention are solved bythe subject matter of the independent claims. Preferred embodiments maybe taken from the dependent claims.

Furthermore, these and other problems underlying the instant inventionare solved by the subject matter of the aspects and embodiments of theinvention outlined in the following in more detail.

SUMMARY Embodiment 1

A beta-herpesvirus, preferably a recombinant beta-herpesvirus, whereinthe beta-herpesvirus comprises at least one heterologous nucleic acid,wherein the at least one heterologous nucleic acid comprises a geneencoding a cellular ligand.

Embodiment 2

The beta-herpesvirus according to embodiment 1, wherein thebeta-herpesvirus is not a murine cytomegalovirus comprising at least oneheterologous nucleic acid, wherein the at least one heterologous nucleicacid comprises a gene encoding a cellular ligand, wherein the cellularligand is RAE1γ, and wherein the beta-herpesvirus is NOT a murinecytomegalovirus deficient in at least one gene product encoded by animmune modulatory gene, wherein the immune modulatory gene is m152.

Embodiment 3

The beta-herpesvirus according to embodiments 1 and 2, wherein thecellular ligand is capable of binding a receptor for the cellularligand.

Embodiment 4

The beta-herpesvirus according to embodiment 3, wherein the receptor forthe cellular ligand is present on the surface of at least one immunecell.

Embodiment 5

The beta-herpesvirus according to any one of embodiments 3 to 4, whereinthe receptor for the cellular ligand is an activating receptor.

Embodiment 6

The beta-herpesvirus according to any one of embodiments 4 to 5, whereinthe at least one immune cell is selected from the group comprising NKcells, γδ T cells and activated CD8⁺ T cells.

Embodiment 7

The beta-herpesvirus according to any one of embodiments 4 to 6, whereinthe at least one immune cell is selected from the group consisting of NKcells, γδ T cells and activated CD8⁺ T cells.

Embodiment 8

The beta-herpesvirus according to any one of embodiments 4 to 7, whereinthe receptor for the cellular ligand is present on the surface of NKcells.

Embodiment 9

The beta-herpesvirus according to any one of embodiments 4 to 8, whereinthe receptor for the cellular ligand is present on the surface of NKcells, γδ T cells and activated CD8⁺ T cells.

Embodiment 10

The beta-herpesvirus according to any one of embodiments 1 to 9, whereinthe cellular ligand is a protein anchored to or in a membrane of a cell,preferably a cell infected by the beta-herpesvirus, viaglycosylphosphatidylinositol (GPI).

Embodiment 11

The beta-herpesvirus according to any one of embodiments 1 to 9, whereinthe cellular ligand is a transmembrane protein.

Embodiment 12

The beta-herpesvirus according to any one of embodiments 1 to 9, whereinthe cellular ligand comprises at least one immunoglobulin-like domain.

Embodiment 13

The beta-herpesvirus according to any one of embodiments 1 to 9, whereinthe at least one immunoglobulin-like domain is selected from the groupcomprising an α1 domain, an α2 domain and an α3 domain.

Embodiment 14

The beta-herpesvirus according to embodiment 13, wherein the cellularligand comprises an α1 domain and an α2 domain.

Embodiment 15

The beta-herpesvirus according to embodiment 13, wherein the cellularligand comprises an α1 domain, an α2 domain and an α3 domain.

Embodiment 16

The beta-herpesvirus according to any one of embodiments 1 to 15,wherein the cellular ligand is an NKG2D ligand.

Embodiment 17

The beta-herpesvirus according to embodiment 16, wherein the NKG2Dligand is a mammalian NKG2D ligand or a homolog thereof.

Embodiment 18

The beta-herpesvirus according to embodiment 17, wherein the NKG2Dligand is selected from the group comprising murine NKG2D ligand andhuman NKG2D ligand.

Embodiment 19

The beta-herpesvirus according to embodiment 18, wherein the NKG2Dligand is a human NKG2D ligand.

Embodiment 20

The beta-herpesvirus according to embodiment 18, wherein the NKG2Dligand is a murine NKG2D ligand.

Embodiment 21

The beta-herpesvirus according to any one of embodiments 18 and 19,wherein the human NKG2D ligand is selected from the group comprisingUL16 binding proteins and MHC class-1-related protein.

Embodiment 22

The beta-herpesvirus according to embodiment 21, wherein the UL16binding protein is selected from the group comprising ULBP2, ULPB1,ULBP3, ULBP4, ULBP5 and ULBP6.

Embodiment 23

The beta-herpesvirus according to embodiment 22, wherein the UL16binding protein is ULBP2.

Embodiment 24

The beta-herpesvirus according to embodiment 22, wherein the UL16binding protein is ULPB1.

Embodiment 25

The beta-herpesvirus according to embodiment 22, wherein the UL16binding protein is ULBP3.

Embodiment 26

The beta-herpesvirus according to embodiment 22, wherein the UL16binding protein is ULBP4.

Embodiment 27

The beta-herpesvirus according to embodiment 22, wherein the UL16binding protein is ULBP5.

Embodiment 28

The beta-herpesvirus according to embodiment 22, wherein the UL16binding protein is ULBP6.

Embodiment 29

The beta-herpesvirus according to embodiment 21, wherein the MHCclass-1-related protein is selected from the group comprising MICA andMICB.

Embodiment 30

The beta-herpesvirus according to embodiment 29, wherein the MHCclass-1-related protein is MICA.

Embodiment 31

The beta-herpesvirus according to embodiment 29, wherein the MHCclass-1-related protein is MICB

Embodiment 32

The beta-herpesvirus according to any one of embodiments 18 and 20,wherein the murine NKG2D ligand is selected from the group comprisingRAE-1 protein, H60 protein and murine UL16 protein-like transcriptprotein.

Embodiment 33

The beta-herpesvirus according to embodiment 32, wherein the RAE-1protein is selected from the group comprising RAE-1α, RAE-1β, RAE-1γ,RAE-1δ, and RAE-1-ε.

Embodiment 34

The beta-herpesvirus according to embodiment 33, wherein the RAE-1protein is RAE-1γ.

Embodiment 35

The beta-herpesvirus according to embodiment 33, wherein the RAE-1protein is RAE-1α.

Embodiment 36

The beta-herpesvirus according to embodiment 33, wherein the RAE-1protein is RAE-1β.

Embodiment 37

The beta-herpesvirus according to embodiment 33, wherein the RAE-1protein is RAE-1δ.

Embodiment 38

The beta-herpesvirus according to embodiment 33, wherein the RAE-1protein is RAE-1ε.

Embodiment 39

The beta-herpesvirus according to embodiment 33, wherein the murine UL16protein-like transcript protein is MULT-1.

Embodiment 40

The beta-herpesvirus according to embodiment 33, wherein the H60 proteinis selected from the group comprising H60a, H60b and H60c.

Embodiment 41

The beta-herpesvirus according to embodiment 40, wherein the H60 proteinis H60a.

Embodiment 42

The beta-herpesvirus according to embodiment 40, wherein the H60 proteinis H60b.

Embodiment 43

The beta-herpesvirus according to embodiment 40, wherein the H60 proteinis H60c.

Embodiment 44

The beta-herpesvirus according to any one of embodiments 3 to 43,wherein the binding of the cellular ligand and the receptor for thecellular ligand is capable of activating NK cells.

Embodiment 45

The beta-herpesvirus according to any one of embodiments 1 to 44,wherein the beta-herpesvirus is suitable for inducing an immune responseagainst a beta-herpesvirus.

Embodiment 46

The beta-herpesvirus according to embodiment 45, wherein the immuneresponse comprises neutralizing antibodies against beta-herpesvirusand/or CD4⁺ T-cells directed against epitopes of beta-herpesvirus and/orCD8⁺ T-cells directed against epitopes of beta-herpesvirus.

Embodiment 47

The beta-herpesvirus according to any one of embodiments 1 to 46,wherein the beta-herpesvirus has a tropism like a wild typebeta-herpesvirus.

Embodiment 48

The beta-herpesvirus according to any one of embodiments 1 to 47,wherein the beta-herpesvirus is capable of infecting professionalantigen presenting cells.

Embodiment 49

The beta-herpesvirus according to any one of embodiments 1 to 48,wherein the beta-herpesvirus is capable of infecting dendritic cells andmacrophages.

Embodiment 50

The beta-herpesvirus according to any one of embodiments 1 to 49,wherein the beta-herpesvirus is capable of infecting professionalantigen presenting cells only.

Embodiment 51

The beta-herpesvirus according to any one of embodiments 1 to 50,wherein the beta-herpesvirus is capable of infecting fibroblasts.

Embodiment 52

The beta-herpesvirus according to any one of embodiments 1 to 51,wherein the beta-herpesvirus is a human beta-herpesvirus.

Embodiment 53

The beta-herpesvirus according to any one of embodiments 1 to 52,wherein the beta-herpesvirus is a cytomegalovirus.

Embodiment 54

The beta-herpesvirus according to embodiment 53, wherein thebeta-herpesvirus is a cytomegalovirus is selected from the groupcomprising human cytomegalovirus, rhesus cytomegalovirus and mousecytomegalovirus.

Embodiment 55

The beta-herpesvirus according to embodiment 54, wherein thebeta-herpesvirus is human cytomegalovirus.

Embodiment 56

The beta-herpesvirus according to embodiment 55, wherein the humancytomegalovirus is a human cytomegalovirus derived from a BacterialArtificial Chromosome.

Embodiment 57

The beta-herpesvirus according to embodiment 56, wherein the BacterialArtificial Chromosome is selected from the group comprising AD169-BAC,modified AD169-BAC, TB40E-BAC, VHLE, Toledo-BAC, Toledo-BAC, TR-BAC,FIX-BAC and transgenic Merlin-BAC.

Embodiment 58

The beta-herpesvirus according to any one of embodiments 1 to 57,wherein the beta-herpesvirus is deficient in at least one gene productencoded by an immune modulatory gene.

Embodiment 59

The beta-herpesvirus according to embodiment 58, wherein thebeta-herpesvirus comprises a deletion of the coding sequence of the atleast one immune modulatory gene.

Embodiment 60

The beta-herpesvirus according to any one of embodiments 58 to 59;wherein the at least one gene product encoded by an immune modulatorygene is a gene product regulating NK cell response.

Embodiment 61

The beta-herpesvirus according to embodiment 60; wherein the geneproduct regulating NK cell response is capable of binding the cellularligand.

Embodiment 62

The beta-herpesvirus according to embodiment 61, wherein the binding ofthe gene product regulating NK cell response and the cellular ligandresults in reduction of expression of the cellular ligand.

Embodiment 63

The beta-herpesvirus according to embodiment 62, wherein the expressionof the cellular ligand is an expression on the surface of a cellinfected with the beta-herpesvirus.

Embodiment 64

The beta-herpesvirus according to any one of embodiments 58 to 63,wherein the at least one immune modulatory gene is selected from thegroup comprising UL16, UL142, m152, m155, m145 and m138.

Embodiment 65

The beta-herpesvirus according to embodiment 64, wherein the immunemodulatory gene is UL16.

Embodiment 66

The beta-herpesvirus according to embodiment 64, wherein the immunemodulatory gene is UL142.

Embodiment 67

The beta-herpesvirus according to embodiment 64, wherein the immunemodulatory gene is m152.

Embodiment 68

The beta-herpesvirus according to embodiment 64, wherein the immunemodulatory gene is m155.

Embodiment 69

The beta-herpesvirus according to embodiment 64, wherein the immunemodulatory gene is m145.

Embodiment 70

The beta-herpesvirus according to embodiment 64, wherein the immunemodulatory gene is m138.

Embodiment 71

The beta-herpesvirus according to any one of embodiments 58 to 65,wherein the at least one immune modulatory gene is UL16 and wherein thecellular ligand is ULBP2.

Embodiment 72

The beta-herpesvirus according to any one of embodiments 58 to 65,wherein the at least one immune modulatory gene is UL16 and wherein thecellular ligand is MICB.

Embodiment 73

The beta-herpesvirus according to any one of embodiments 58 to 65,wherein the at least one immune modulatory gene is UL16 and wherein thecellular ligand is ULBP1.

Embodiment 74

The beta-herpesvirus according to any one of embodiments 58 to 65,wherein the at least one immune modulatory gene is UL16 and wherein thecellular ligand is ULBP6.

Embodiment 75

The beta-herpesvirus according to any one of embodiments 58 to 64 and65, wherein the at least one immune modulatory gene is UL142 and whereinthe cellular ligand is MICA.

Embodiment 76

The beta-herpesvirus according to any one of embodiments 58 to 64 and67, wherein the at least one immune modulatory gene is m152 and whereinthe cellular ligand is selected from the group comprising RAE-1α,RAE-1β, RAE-1γ, RAE-1δ, and RAE-1-ε.

Embodiment 77

The beta-herpesvirus according to any one of embodiments 58 to 64 and68, wherein the at least one immune modulatory gene is m155 and whereinthe cellular ligand is selected from the group comprising H60a, H60b andH60c.

Embodiment 78

The beta-herpesvirus according to any one of embodiments 58 to 64 and69, wherein the at least one immune modulatory gene is m145 and whereinthe cellular ligand is MULT-1.

Embodiment 79

The beta-herpesvirus according to any one of embodiments 58 to 64 and70, wherein the at least one immune modulatory gene is m138 and whereinthe cellular ligand is MULT-1.

Embodiment 80

The beta-herpesvirus according to any one of embodiments 58 to 64 and70, wherein the at least one immune modulatory gene is m138 and whereinthe cellular ligand is selected from the group comprising H60a, H60b andH60c.

Embodiment 81

The beta-herpesvirus according to any one of embodiments 58 to 64 and70, wherein the at least one immune modulatory gene is m138 and whereinthe cellular ligand is selected from the group comprising RAE-1α,RAE-1β, RAE-1γ, RAE-1δ, and RAE-1-ε.

Embodiment 82

The beta-herpesvirus according to any one of embodiments 58 to 81,wherein the beta-herpesvirus is deficient in one or more additional geneproduct(s) each encoded by an additional immune modulatory gene.

Embodiment 83

The beta-herpesvirus according to any one of embodiments 58 to 82,preferably embodiment 82, wherein the beta-herpesvirus comprises adeletion of the coding sequence of the additional immune modulatorygene.

Embodiment 84

The beta-herpesvirus according to any one of embodiments 81 to 82;wherein the at least one additional gene product encoded by theadditional immune modulatory gene is a gene product regulating NK cellresponse encoded by an immune modulatory gene selected from the groupcomprising UL16, UL18, UL40, UL142, m152, m155, m145 and m138.

Embodiment 85

The beta-herpesvirus according to any one of embodiments 81 to 83wherein the at least one additional gene product encoded by theadditional immune modulatory gene is a gene product regulating MHC classI presentation.

Embodiment 86

The beta-herpesvirus according to embodiment 85, wherein the geneproduct regulating MHC class I presentation is a gene product encoded byan immune modulatory gene selected from the group comprising US6, US3,US2 and US11.

Embodiment 87

The beta-herpesvirus according to any one of embodiments 1 to 86,wherein the beta-herpesvirus comprises the deletion of at least onemiRNA.

Embodiment 88

The beta-herpesvirus according to embodiment 87, wherein the miRNA iscapable of binding a transcript of the cellular ligand.

Embodiment 89

The beta-herpesvirus according to any one of embodiments 87 to 88,preferably embodiment 88, wherein the miRNA is capable of repressing thetranslation of the gene coding for the cellular ligand.

Embodiment 90

The beta-herpesvirus according to any one of embodiments 87 to 89,preferably embodiment 89, wherein the miRNA is miRNA-UL112.

Embodiment 91

The beta-herpesvirus according to any one of embodiments 1 to 90,wherein the beta-herpesvirus is deficient in at least one gene productencoded by a gene regulating viral replication.

Embodiment 92

The beta-herpesvirus according to any one of embodiments 1 to 91,wherein the gene regulating viral replication is selected from the groupcomprising IE1, pp 71 and pp 65.

Embodiment 93

The beta-herpesvirus according to any one of embodiments 1 to 92,wherein the beta-herpesvirus is deficient in at least one gene productencoded by an essential gene.

Embodiment 94

The beta-herpesvirus according to embodiment 93, wherein the essentialgene is selected from the group comprising UL32, UL34, UL37.1, UL44,UL46, UL48, UL48, UL49, UL50, UL51, UL52, UL53, UL54, UL55, UL56, UL57,UL60, UL70, UL71, UL73, UL75, UL76, UL77, UL79, UL80, UL84, UL85, UL86,UL87, UL89.1, UL90, UL91, UL92, UL93, UL94, UL95, UL96, UL98, UL99,UL100, UL102, UL104, UL105, UL115 and UL122.

Embodiment 95

The beta-herpesvirus according to any one of embodiments 1 to 93,wherein the beta-herpesvirus is deficient in at least one glycoprotein.

Embodiment 96

The beta-herpesvirus according to embodiment 95, wherein theglycoprotein is selected from the group comprising gB.

Embodiment 97

The beta-herpesvirus according to any one of embodiments 1 to 96,wherein the beta-herpesvirus encodes at least one additionalheterologous nucleic acid.

Embodiment 98

The beta-herpesvirus according to embodiment 97, wherein the at leastone additional heterologous nucleic acid is a functional nucleic acid,preferably the functional nucleic acid is selected from the groupcomprising antisense molecules, ribozymes and RNA interference mediatingnucleic acids.

Embodiment 99

The beta-herpesvirus according to embodiment 97, wherein the at leastone additional heterologous nucleic acid is a heterologous nucleic acidcoding for a peptide, oligopeptide, polypeptide or protein.

Embodiment 100

The beta-herpesvirus according to embodiment 99, wherein the peptide,oligopeptide, polypeptide or protein constitutes or comprises at leastone antigen.

Embodiment 101

The beta-herpesvirus according to embodiment 100, wherein the antigen isan antigen selected from the group comprising tumor antigens, tumorassociated antigens, viral antigens, bacterial antigens and parasiteantigens.

Embodiment 102

The beta-herpesvirus according to embodiment 101, wherein the viralantigen is an antigen derived from a virus, wherein the virus isselected from the group comprising HIV, Influenza, HPV and RSV.

Embodiment 103

The beta-herpesvirus according to embodiment 102, wherein the viralantigen from Influenza is selected from the group comprisinghaemaglutinin.

Embodiment 104

The beta-herpesvirus according to embodiment 103, wherein thehaemaglutinin is selected from the group comprising haemaglutininfull-length form and haemaglutinin headless form.

Embodiment 105

The beta-herpesvirus according to embodiment 102, wherein the viralantigen from RSV is selected from the group comprising glycoprotein Fand glycoprotein G.

Embodiment 106

The beta-herpesvirus according to embodiment 102, wherein the viralantigen from HIV is selected from the group comprising HIV-1 gag.

Embodiment 107

The beta-herpesvirus according to embodiment 102, wherein the viralantigen from HPV is selected from the group comprising E6 29-38, E629-37, E6 31-38, E6 52-61, E6 and E7.

Embodiment 108

The beta-herpesvirus according to embodiment 101, wherein the bacterialantigen is an antigen derived from a bacterium, wherein the bacterium isselected from the group comprising mycobacterium, Helicobacter pyloriand Listeria.

Embodiment 109

The beta-herpesvirus according to embodiment 108, wherein the bacterialantigen from Helicobacter pylori is selected from the group comprisingurease, VacA, CagA, heat shock protein, neutrophil-activating proteinouter membrane lipoprotein and babA2.

Embodiment 110

The beta-herpesvirus according to embodiment 108, wherein mycobacteriumis selected from the group comprising Mycobacterium tuberculosis.

Embodiment 111

The beta-herpesvirus according to embodiment 110, wherein the bacterialantigen from Mycobacterium tuberculosis is selected from the groupcomprising Antigen 85 A, Antigen 85B and Antigen 85B-TB10.4.

Embodiment 112

The beta-herpesvirus according to embodiment 108, wherein Listeria isselected from the group comprising Listeria monocytogenes.

Embodiment 113

The beta-herpesvirus according to embodiment 108, wherein the bacterialantigen from Listeria is selected from the group comprisinglisteriolysin O (LLO).

Embodiment 114

The beta-herpesvirus according to embodiment 101, wherein the parasiteantigen is an antigen derived from a parasite, wherein the parasite isselected from the group comprising Plasmodium.

Embodiment 115

The beta-herpesvirus according to embodiment 114, wherein Plasmodium isselected from the group comprising Plasmodium falciparum, Plasmodiumvivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi.

Embodiment 116

The beta-herpesvirus according to embodiment 115, wherein Plasmodium isPlasmodium falciparum.

Embodiment 117

The beta-herpesvirus according to embodiment 116, wherein the parasiteantigen from Plasmodium falciparum is selected from the groupcircumsporozoite protein.

Embodiment 118

The beta-herpesvirus according to any one of embodiments 1 to 117 for orsuitable for use in a method for the treatment of a subject and/or foruse in a method for the vaccination of a subject.

Embodiment 119

The beta-herpesvirus according to embodiment 118, wherein the subject isa mammal, preferably a human being.

Embodiment 120

The beta-herpesvirus according to any one of embodiments 118 to 119,wherein the subject is suffering from a disease or is at risk ofsuffering from a disease.

Embodiment 121

The beta-herpesvirus according to any one of embodiments 118 to 120,wherein the vaccination is a vaccination against a disease.

Embodiment 122

The beta-herpesvirus according to any one of embodiments 120 to 121,wherein the disease is a disease or condition which is associated withbeta-herpesvirus infection, preferably human cytomegalovirus infection.

Embodiment 123

The beta-herpesvirus according to any one of embodiments 120 to 122,wherein the disease or condition is selected from the group comprisingcongenital inclusion disease.

Embodiment 124

The beta-herpesvirus according to any one of embodiments 118 to 123,wherein the subject is a pregnant female or female of reproductive age,preferably a pregnant human or a human of reproductive age.

Embodiment 125

The beta-herpesvirus according to any one of embodiments 118 to 124,wherein the treatment is or is suitable for or capable of preventing thetransfer of a beta-herpesvirus, preferably human cytomegalovirus, fromthe female to a fetus and/or to an embryo carried by the female or to becarried in the future by the female.

Embodiment 126

The beta-herpesvirus according to any one of embodiments 118 to 125,wherein the treatment is for or is suitable for the generation of orcapable of generating an immune response in the female body, wherebypreferably such immune response confers protection to a fetus and/or toan embryo carried or to be carried in the future by the female againstbeta-herpesvirus and/or a disease or condition associated withbeta-herpesvirus infection,

Embodiment 127

The beta-herpesvirus according to embodiment 126, wherein thebeta-herpesvirus is a cytomegalovirus, preferably human cytomegalovirus.

Embodiment 128

The beta-herpesvirus according to any one of embodiments 1 to 127,wherein the beta-herpesvirus is suitable of inducing an immune responseagainst a beta-herpesvirus.

Embodiment 129

The beta-herpesvirus according to embodiment 128, wherein the immuneresponse comprises neutralizing antibodies against beta-herpesvirusand/or CD4⁺ T-cells directed against epitopes of beta-herpesvirus and/orCD8⁺ T-cells directed against epitopes of beta-herpesvirus.

Embodiment 130

The beta-herpesvirus according to any one of embodiments 120 to 121,wherein the disease is a disease selected from the group comprisingbacterial disease, viral disease, parasite disease and tumors.

Embodiment 131

The beta-herpesvirus according to embodiment 130, wherein the viraldisease is selected from the group comprising AIDS, Influenza and RSVinfection

Embodiment 132

The beta-herpesvirus according to embodiment 130, wherein the bacterialdisease is selected from the group comprising Tuberculosis andlisteriosis.

Embodiment 133

The beta-herpesvirus according to embodiment 130, wherein the parasitedisease is selected from the group comprising Malaria.

Embodiment 134

A beta-herpesvirus for use in a method for the treatment and/orprevention of HIV infection,

wherein the beta-herpesvirus expresses a cellular ligand and an antigen,wherein the cellular ligand is selected from the group comprising ULBP2,ULPB1, ULBP3, ULBP4, ULBP5, ULBP6, MICA, MICB, RAE-1α, RAE-1β, RAE-1γ,RAE-1δ, RAE-1-ε, MULT-1, H60a, H60b and H60c,wherein the antigen is selected from the group comprising HIV-1 gag, andwherein the beta-herpesvirus preferably is a beta-herpesvirus accordingto any one of embodiments 1 to 133.

Embodiment 135

A beta-herpesvirus for use in a method for the treatment and/orprevention of influenza virus infection,

wherein the beta-herpesvirus expresses a cellular ligand and an antigen,wherein the cellular ligand is selected from the group comprising ULBP2,ULPB1, ULBP3, ULBP4, ULBP5, ULBP6, MICA, MICB, RAE-1α, RAE-1β, RAE-1γ,RAE-1δ, RAE-1-ε, MULT-1, H60a, H60b and H60c,wherein the antigen is selected from the group comprising haemaglutininfull-length form and haemaglutinin headless form, andwherein the beta-herpesvirus preferably is a beta-herpesvirus accordingto any one of embodiments 1 to 133.

Embodiment 136

A beta-herpesvirus for use in a method for the treatment and/orprevention of HPV infection,

wherein the beta-herpesvirus expresses a cellular ligand and an antigen,wherein the cellular ligand is selected from the group comprising ULBP2,ULPB1, ULBP3, ULBP4, ULBP5, ULBP6, MICA, MICB, RAE-1α, RAE-1β, RAE-1γ,RAE-1δ, RAE-1-ε, MULT-1, H60a, H60b and H60c,wherein the antigen is selected from the group comprising E6 29-38, E629-37, E6 31-38, E6 52-61, E6 and E7, andwherein the beta-herpesvirus preferably is a beta-herpesvirus accordingto any one of embodiments 1 to 133.

Embodiment 137

A beta-herpesvirus for use in a method for the treatment and/orprevention of RSV infection,

wherein the beta-herpesvirus expresses a cellular ligand and an antigen,wherein the cellular ligand is selected from the group comprising ULBP2,ULPB1, ULBP3, ULBP4, ULBP5, ULBP6, MICA, MICB, RAE-1α, RAE-1β, RAE-1γ,RAE-1δ, RAE-1-ε, MULT-1, H60a, H60b and H60c,wherein the antigen is selected from the group comprising glycoprotein Fand glycoprotein G, andwherein the beta-herpesvirus preferably is a beta-herpesvirus accordingto any one of embodiments 1 to 133.

Embodiment 138

A beta-herpesvirus for use in a method for the treatment and/orprevention of Helicobacter pylori infection,

wherein the beta-herpesvirus expresses a cellular ligand and an antigen,wherein the cellular ligand is selected from the group comprising ULBP2,ULPB1, ULBP3, ULBP4, ULBP5, ULBP6, MICA, MICB, RAE-1α, RAE-1β, RAE-1γ,RAE-1δ, RAE-1-ε, MULT-1, H60a, H60b and H60c,wherein the antigen is selected from the group comprising urease, VacA,CagA, heat shock protein, neutrophil-activating protein outer membranelipoprotein and babA2, and wherein the beta-herpesvirus preferably is abeta-herpesvirus according to any one of embodiments 1 to 133.

Embodiment 139

A beta-herpesvirus for use in a method for the treatment and/orprevention of Mycobacterium tuberculosis infection,

wherein the beta-herpesvirus expresses a cellular ligand and an antigen,wherein the cellular ligand is selected from the group comprising ULBP2,ULPB1, ULBP3, ULBP4, ULBP5, ULBP6, MICA, MICB, RAE-1α, RAE-1β, RAE-1γ,RAE-1δ, RAE-1-ε, MULT-1, H60a, H60b and H60c,wherein the antigen is selected from the group comprising Antigen 85 A,Antigen 85B and Antigen 85B-TB10.4, andwherein the beta-herpesvirus preferably is a beta-herpesvirus accordingto any one of embodiments 1 to 133.

Embodiment 140

A beta-herpesvirus for use in a method for the treatment and/orprevention of Listeria infection, preferably Listeria monocytogenesinfection,

wherein the beta-herpesvirus expresses a cellular ligand and an antigen,wherein the cellular ligand is selected from the group comprising ULBP2,ULPB1, ULBP3, ULBP4, ULBP5, ULBP6, MICA, MICB, RAE-1α, RAE-1β, RAE-1γ,RAE-1δ, RAE-1-ε, MULT-1, H60a, H60b and H60c,wherein the antigen is selected from the group comprising listeriolysinO (LLO), and wherein the beta-herpesvirus preferably is abeta-herpesvirus according to any one of embodiments 1 to 133.

Embodiment 141

A beta-herpesvirus for use in a method for the treatment and/orprevention of Plasmodium falciparum infection,

wherein the beta-herpesvirus expresses a cellular ligand and an antigen,wherein the cellular ligand is selected from the group comprising ULBP2,ULPB1, ULBP3, ULBP4, ULBP5, ULBP6, MICA, MICB, RAE-1α, RAE-1β, RAE-1γ,RAE-1δ, RAE-1-ε, MULT-1, H60a, H60b and H60c,wherein the antigen is selected from the group comprisingcircumsporozoite protein, andwherein the beta-herpesvirus preferably is a beta-herpesvirus accordingto any one of embodiments 1 to 133.

Embodiment 142

Use of a beta-herpesvirus as defined in any one of embodiments 1 to 141for the manufacture of a medicament.

Embodiment 143

Use of a beta-herpesvirus according to embodiment 142, wherein themedicament is for the treatment and/or prevention of beta-herpesvirusinfection.

Embodiment 144

Use of a beta-herpesvirus according to any one of embodiment 142 to 143,wherein the medicament is for the treatment and/or prevention of adisease or condition associated with beta-herpesvirus infection,preferably human cytomegalovirus infection.

Embodiment 145

Use of a beta-herpesvirus according to any one of embodiment 1 to 141,for the manufacture of a vaccine.

Embodiment 146

Use of a beta-herpesvirus according to embodiment 145, wherein thevaccine is for the treatment and/or prevention of a disease.

Embodiment 147

Use of a beta-herpesvirus according to embodiment 146, wherein thedisease is beta-herpesvirus infection.

Embodiment 148

Use of a beta-herpesvirus according to any one of embodiments 146 and147, wherein the disease is a disease as defined in any one ofembodiments 130 to 133.

Embodiment 149

Use of a beta-herpesvirus according to embodiment 145, wherein thevaccine is for the treatment and/or prevention of a disease or conditionassociated with beta-herpesvirus infection, preferably humancytomegalovirus infection.

Embodiment 150

Use of a beta-herpesvirus according to embodiment 149, wherein thevaccine is or is suitable for the administration to a subject, wherebythe subject is selected form the group comprising a pregnant female, afemale of reproductive age, a donor of a transplant, a recipient of atransplant and a subject being infected with HIV or being at risk ofbeing infected with HIV.

Embodiment 151

Use of a beta-herpesvirus according to embodiment 150, wherein the donoris a potential donor and/or the recipient is a potential recipient.

Embodiment 152

A nucleic acid coding for a beta-herpesvirus as defined in any of thepreceding embodiments.

Embodiment 153

A vector, preferably an expression vector, comprising the nucleic acidaccording to embodiment 152.

Embodiment 154

A host cell comprising a nucleic acid according to embodiment 152 or avector according to embodiment 153.

Embodiment 155

A pharmaceutical composition comprising a beta-herpesvirus according toany one of the preceding embodiments, a nucleic acid of embodiment 152and/or a vector of embodiment 153, and a pharmaceutically acceptablecarrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic illustration of the cloning process and genomeorganization of RAE-1γMCMV; FIG. 1B is a series of histograms showingthe result of FACS analysis of the surface expression of RAE-1γ in MCMVinfected cells; FIG. 1C is a diagram indicating virus load in differentorgans of infected mice with and without blocking anti-NKG2D monoclonalantibody treatment; FIG. 1D is a diagram indicating virus load insalivary glands at different time points; FIG. 1E is a is a diagramindicating copies of viral genome in different organs of infected miceat different time points.

FIG. 2A is a diagram indicating virus load in different organs ofinfected neonatal mice at different time points; FIG. 2B is a diagramindicating copies of MCMV genome in different organs of infectedneonatal mice at different time points.

FIG. 3A is a diagram indicating the percentage of tetramer-specific CD8⁺T cells of mice infected with MCMV at different time points; FIG. 3B isa bar diagram indicating the percentage of IE1/m123-specific CD8⁺ Tcells of mice infected with MCMV expressing various cell surfacemolecules; FIG. 3C is a histogram showing the result of FACS analysis ofsurface expression of NKG2D on IE1/m123-specific CD8⁺ T cells in spleenof mice infected with MCMV; FIG. 3D is a series of dot plots showing theresult of FACS analysis of tetramer-positive and/or IFN-γ positivesplenocytes of mice infected with MCMV; FIG. 3E is a series of dot plotsshowing the result of FACS analysis of splenocytes of infected micestimulated in the presence of the αCD107a antibody and co-stained forIFN-γ and TNF-α production.

FIG. 4A is a diagram indicating the virus titer in spleen of BALB/c miceinfected with MCMV after transfer of memory CD8⁺ T from latentlyinfected μMT/μMT B cell-deficient mice; FIG. 4B are diagrams indicatingthe percentage of IE1/m123 MHC class I tetramer per CD8⁺ T cells invarious organs of mice challenged with of salivary gland derived MCMV;FIG. 4C is a survival curve indicating survival of different vaccinatedmice as a function of time.

FIG. 5A is a diagram indicating viral load of various organs in latentlyinfected B cell-deficient mice depleted of CD4⁺, CD8⁺, and NK1.1⁺ cells;FIG. 5B is a series of histograms showing the result of FACS analysis ofsurface RAE-1γ expression of SVEC4-10 cells infected with recurrentplaque purified RAE-1γMCMV viruses; FIG. 5C is a diagram indicating thevirus titer in spleen of mice infected with MCMV treated with or withoutblocking anti-NKG2D antibody.

FIG. 6A is a survivorship curve indicating survival of IFNa/bR ko miceinfected with MCMV as a function of time; FIG. 6B is a diagramindicating the viral load of organs of γ-irradiated infected miceoptionally depleted for NK cells.

FIG. 7A depicts diagrams indicating antiviral antibody titers in serumof infected females and neonates and an illustrative scheme of theexperimental protocol; FIG. 7B is a diagram indicating viral load invarious organs of infected neonates and an illustrative scheme of theexperimental protocol.

FIG. 8A is a diagram indicating virus titer of MCMV in vitro as afunction over time; FIG. 8B is a series of histograms showing the resultof FACS analysis of the surface expression of NKG2D ligands on infectedcells.

FIG. 9A depicts diagrams indicating the virus load in organs of infectedmice treated with or without with blocking anti-NKG2D antibody; FIG. 9Bis a diagram indicating the percentage of IFN⁺CD8⁺ T cells ofpeptide-stimulated splenocytes from mice infected with MCMV.

FIG. 10 depicts diagrams indicating the viral load in organs of MCMVinfected neonatal BALB/c mice.

FIG. 11 is a series of dot plots showing the results of FACS analysis ofthe percentage of CD11b cDCs and CD8α cDCs in infected mice.

FIG. 12A is a schematic illustration of the cloning process and genomeorganization of RAE-1γMCMV and RAE-1γMCMVList; FIG. 12B is anillustrative scheme of the experimental protocol for experimentsapplying Listeria monocytogenes challenge.

FIG. 13 is a diagram indicating virus titer in vitro as a function overtime.

FIG. 14 are diagrams indicating virus titer in salivary glands ofinfected mice.

FIGS. 15A to 15B are diagrams indicating percentage of IFN-γ⁺ CD8⁺ Tcells of peptide-stimulated splenocytes from infected mice.

FIG. 16A depicts diagrams indicating CFU of listeria monocytogenes afterchallenge of virus infected mice; FIG. 16B is a series of micrographsillustrating the effect of vaccination by Rae-1γ MCMVList in limitingdepletion of T cells from PALS in spleen.

FIG. 17 depicts diagrams indicating the percentage of IFNγ⁺ CD8⁺ T cellsof splenocytes from vaccinated mice after challenge with Listeriamonocytogenes.

FIG. 18 depicts diagrams indicating CFU of listeria monocytogenes ofvaccinated mice challenged with Listeria monocytogenes after depletionfor CD8⁺ T cells.

FIG. 19 is a series of micrographs indicating liver pathology afterListeria monocytogenes challenge in vaccinated mice.

FIG. 20 depicts diagrams indicating the percentage of IFNγ⁺ NK cells andpercentage of total NK cells of splenocytes of vaccinated mice afterchallenge with Listeria monocytogenes.

FIG. 21 depicts diagrams indicating the frequency of CD8α⁺ DCs(CD11c^(hi) CD8+⁺) and CD11b+ DCs (CD11c^(hi) CD8α⁻) of infected mice.

FIG. 22 depicts diagrams indicating the frequency of pDC and theconcentration of serum IFNα of infected mice.

FIG. 23 depicts diagrams indicating the total number of CD8⁺ T cells,effector memory CD8⁺ T cells and virus specific CD8⁺ T cells infectedmice.

FIG. 24 A to E are schematic illustrations of the genome organizationand cloning process of Rae-1γMCMV or WT MCMV expressing HA andHA-headless expressed.

FIG. 25 is a diagram indicating virus titer in vitro as a function overtime.

FIG. 26 is a schematic illustration of the genome organization andcloning process of GAPINSATAM expressing MCMV.

FIGS. 27A and B depict diagrams indicating the percentage of IFNγ⁺ CD8⁺T cells as a result of peptide-stimulation of splenocytes from infectedmice.

FIG. 28 is a diagram indicating viral load of various organs of infectedmice.

FIG. 29 is a diagram indicating the percentage of IFNγ⁺ CD8⁺ T cells ofsplenocytes of infected mice.

FIGS. 30 A and B are schematic illustrations of the genome organizationand cloning process of ULBP2 expressing HCMV; FIG. 30 C is a series ofWestern blots showing the expression of ULBP2 in infected cells; FIG.30D is a histogram showing the surface expression of ULBP2 on humanforeskin fibroblast infected with HCMV TB40 or HCMV TB40 expressingULBP2; FIG. 30 E is a diagram showing the results of NK cellcytotoxicity using uninfected human foreskin fibroblast or cellsinfected with either HCMV TB40 or HCMV TB40 expressing ULBP2.

FIG. 31 is a schematic illustration of the cloning process of an HCMVvaccine expressing ULBP2 and influenza HA.

FIG. 32A are diagrams indicating the percentage of survival and bodyweight loss (upper panel), and CFU of listeria monocytogenes (lowerpanel) of virus infected Balb/c mice after challenge with L.monocytogenes; FIG. 32B is a survivorship curve indicating survival ofvirus infected Balb/c mice after challenge with L. monocytogenes; FIG.32C is a diagram showing the percentage of LLO-specific CD8+ Tcell-mediated cytotoxicity.

FIG. 33A is a diagram indicating viral load of lungs in Balb/c miceinfected with MCMV; FIG. 33 B is a diagram showing the percentage ofLLO-specific CD8⁺ T cells in lungs of Balb/c mice infected with MCMV.

FIG. 34A is a diagram indicating viral load of spleen in Balb/c miceinfected with MCMV and treated with NKG2D blocking antibody; FIG. 34 Bis a diagram indicating the absolute number of LLO-specific CD8⁺ T cellsin Balb/c mice infected with MCMV and treated with NKG2D blockingantibody.

FIG. 35A is a diagram showing the percentage of BrdU⁺ total CD8⁺ T cellsin BALB/c mice infected with MCMV and injected with BrdU; FIG. 35B is adiagram showing the percentage LLO-specific CD8⁺ T cells in Balb/c miceinfected with MCMV and injected with BrdU; FIG. 35C is a diagram showingthe percentage of BrdU⁺ LLO-specific CD8⁺ T cells in Balb/c miceinfected with MCMV and injected with BrdU.

FIG. 36 is a diagram indicating virus titer of WT-MCMV, MCMV-SIINFEKLand RAE-1γMCMV-SIINFEKL in vitro as a function over time.

FIG. 37 is a series of diagrams showing the percentage of IFNγ⁺ CD8⁺ Tcells in C57BL/6 mice infected f.p. with MCMV.

FIG. 38 is a series of diagrams showing the percentage of IFNγ⁺ CD8⁺ Tcells in C57BL/6 mice infected i.v. with MCMV.

FIG. 39 is a series of diagrams indicating CFU of listeria monocytogenesof vaccinated mice challenged with Listeria monocytogenes.

FIG. 40 A is a series of diagrams showing the percentage of SIINFEKL-and M45-tetramer-specific CD8⁺ T cells in MCMV infected C57BL/6 mice;FIG. 40 B is a diagram indicating the viral load in lungs of MCMVinfected C57BL/6 mice.

FIG. 41A is a survivorship curve indicating survival of C57BL/6 miceinfected with MCMV and challenged with PR8 virus as a function of time;FIG. 41 B is a weight loss curve indicating the weight loss of C57BL/6mice infected with MCMV and challenged with PR8 virus as a function oftime.

FIG. 42 is a diagram showing the percentage of IFNγ⁺ CD8⁺ T cells inMCMV infected BALB/c mice.

DETAILED DESCRIPTION

The present inventor has surprisingly found that the infection of asubject by a beta-herpesvirus, preferably a recombinantbeta-herpesvirus, wherein a cellular ligand, preferably recognized by anactivating receptor on immune cells, is inserted into the genome of thebeta-herpesvirus, results in eliciting an immune response against thebeta-herpesvirus sufficient to protect against infection with wild typebeta-herpesvirus, although said beta-herpesvirus is severely attenuatedcompared to a respective wild type beta-herpesvirus. Accordingly, saidbeta-herpesvirus was found to provide a safe and protectivebeta-herpesvirus vaccine, suitable for vaccination againstbeta-herpesvirus infection. More specifically, the present inventor hassurprisingly found that the infection of a subject by a CMV, preferablya recombinant CMV, wherein a cellular ligand, preferably recognized byan activating receptor on immune cells, is inserted into the genome ofthe CMV, results in eliciting an immune response against the CMVsufficient to protect against infection with wild type CMV, althoughsaid CMV is severely attenuated compared to a respective wild type CMV.Accordingly, said CMV was found to provide a safe and protective CMVvaccine, suitable for vaccination against CMV infection.

The present inventor surprisingly found that a CMV encoding a ligand forNKG2D provides a safe and protective CMV vaccine, suitable forvaccination against CMV infection.

Furthermore, the present inventor has found that a beta-herpesvirusaccording to the present invention, wherein a nucleic acid coding for acellular ligand and a nucleic acid coding for an antigen and epitope,respectively, of a pathogen, preferably an immunodominant antigen, isinserted into the genome of the beta-herpesvirus, may serve as a vaccineor be used as a vaccine or vaccination vector for vaccination againstinfection with the pathogen. The pathogen is preferably a virus, abacterium or a parasite.

In connection therewith a person skilled in the art will immediatelyacknowledge that immune cells, such as natural killer cells, alsoreferred to herein as NK cells, CD8⁺ T cells and dendritic cells, alsoreferred to herein as DCs, have basically the same function in differentmammalian species, such as mouse and man, as may be taken fromimmunology text books such as “Janeway's Immunobiology”, 8^(th)addition, by Kenneth Murphy.

Furthermore, it has to be acknowledged by a person skilled in the artthat any characteristic feature, embodiment of and any statement madeherein in relation to beta-herpesviruses such as MCMV equally applies toHCMV. Furthermore, it will be acknowledged by a person skilled in theart that the beta-herpesvirus according to the present invention will,in a preferred embodiment, exhibit the following characteristics asobserved for HCMV and MCMV, respectively: multiple infections occur withMCMV in mouse and HCMV in human, respectively, (Boppana, S. B. et al.,2001, N Engl J Med 344:1366-1371; Cicin-Sain, L. et al., 2005, J Virol79:9492-9502); a response of neutralizing antibodies against CMV iscaused by infection with MCMV in mouse and HCMV in human, respectively(Farrell, H. E. and Shellam, G. R. 1990, J Gen Virol 71 (Pt 3):655-664;Farrell, H. E. and Shellam, G. R. 1991, J Gen Virol 72 (Pt 1):149-156;Gerna, G. A. et al., 2008, J Gen Virol 89:853-865); memory inflation,which represents a very characteristic CD8+ T cell response, is causedby infection with MCMV in mouse and HCMV in human, respectively, and hasalmost identical kinetics in both human and mouse (Karrer, U. et al.,2003, J Immunol 170:2022-2029; Karrer, U. et al., 2004, J Virol78:2255-2264; Klenerman, P. and Dunbar, P. R. 2008, Immunity.29:520-522; Komatsu, H. et al., 2003, ClinExp Immunol 134:9-12;Holtappels R. et al., 2002, J Virol 76:151-164; Holtappels R. et al.,2001, J Virol 75:6584-6600; Snyder, C. M. et al., 2008, Immunity;29(4):650-9).

It can thus be immediately taken from the above that infection of a hostwith its corresponding CMV species such as infection of human with HCMVand infection of mouse with MCMV, as well as immune responses to saidinfections in human and mouse, respectively, are comparable. Moreparticularly, numerous publications of the prior art acknowledge theusefulness and appropriateness of the mouse model for studying CMVinfection and immune response to said infection. For example, evidencefor the above is found, among others, in the publications of Reddehaseet al. (Reddehase, M. J. et al., 1985, J Virol 55(2):264-73; Reddehase,M. J., 2002, Nat Rev Immunol 2:831-844), Pollock and Virgin (Pollock, J.L. and Virgin, H. W. 4^(th), J Virol. 1995, 69(3):1762-8), Koffron etal. (Koffron, A. J. et al., 1998, J Virol 72(1):95-103), Brune et al.(Brune, W. et al., 2001, Curr Protoc Immunol., Chapter 19:Unit 19.7.)and Babić et al. (Babić, M. et al., 2011, Trends Mol. Med.17(11):677-85).

Furthermore there is increasing evidence that in connection withinfection of a host with a corresponding CMV species, such as infectionof human with HCMV and infection of mouse with MCMV, basically the sameorgans and tissues are infected, disease preferably occurs inimmunocompromised subjects and the overall immune response caused bysaid infections is similar. Additionally, the value of CD8⁺ T cells inCMV infection, particularly in HCMV infection has been shown. Evidencetherefore may for example be taken from Riddell et al. (Riddell, S. R.et al., 1997, Rev Med Virol 7(3):181-192; Riddell, S. R. et al., 1993,Curr Opin Immunol 5(4):484-91; Riddell, S. R. et al., 1992, Science257(5067):238-41), Riddell and Greenberg (Riddell, S. R. and Greenberg,P. D., 1995, Annu Rev Immunol 13:545-86), Brestrich et al. (Brestrich,G. et al., 2009, J Immunother, 32(9):932-40), Lilleri et al. (Lilleri,D. et al., 2009, J Infect Dis, 199(6):829-36), Kapp et al. (Kapp, M. etal., 2007, Cytotherapy, 9(8):699-711) Wilkinson et al., (Wilkinson, G.W. et al., 2008, J Clin Virol 41(3):206-12), Braud et al. (Braud, V. M.et al., 2002, Curr Top Microbiol Immunol 269:117-29) and Jackson et al.(Jackson, S. E. et al., 2011, Virus Res, 157(2):151-60).

In an embodiment of the beta-herpesvirus according to the presentinvention, wherein the beta-herpesvirus comprises at least oneheterologous nucleic acid and wherein the at least one heterologousnucleic acid comprises a gene encoding a cellular ligand, the at leastone heterologous nucleic acid is a nucleic acid which according to itsnucleotide sequence preferably is not comprised in or part of the genomeof the wild type beta-herpesvirus. In connection therewith it will beunderstood that a beta-herpesvirus according to the present invention inan embodiment comprises the heterologous nucleic acid comprising thegene encoding the cellular ligand as well as a heterologous nucleic acidcomprising one or more than one gene encoding one or more cellularligand(s). In connection therewith, the nucleic acid coding for thecellular ligand is also referred to herein as heterologous nucleic acid.

In an alternative embodiment the beta-herpesvirus according to thepresent invention comprises more than one heterologous nucleic acids.Accordingly, a beta-herpesvirus of the present invention comprises in anembodiment the at least one additional heterologous nucleic acid. In anembodiment said at least one additional heterologous nucleic acid is aheterologous nucleic acid which according to its sequence preferably isnot comprised in or part of the genome of the wild type beta-herpesvirusand more preferably is a functional nucleic acid. Preferably, thefunctional nucleic acid is selected from the group comprising antisensemolecules, ribozymes and RNA interference mediating nucleic acids.Alternatively, at least one additional heterologous nucleic acid is anucleic acid coding for a peptide, oligopeptide, polypeptide or protein,wherein in a more preferred embodiment the peptide, oligopeptide,polypeptide or protein constitutes or comprises at least one antigen andepitope, respectively. Preferably said antigen is an antigen selectedfrom the group comprising tumor antigens, tumor associated antigens,viral antigens, bacterial antigens and parasite antigens.

The term “antigen” as used herein preferably means a foreign molecule orpart of a foreign molecule, such as a protein derived from a bacterium,or a peptide derived from said protein. Said foreign molecule whenintroduced into a body such as a human or mammalian body, is recognizedby the receptor of an immune cell, such as the receptor of an NK cell,and preferably triggers an immune response against said antigen, such asthe production of an antibody directed against said antigen or triggersan effector function of an immune cell.

The term “epitope” as used herein preferably means the distinctmolecular feature of an antigen, preferably a feature on the surface ofan antigen, that is recognized by the immune system, preferably byantibodies, B cells and/or T cells.

The term “homolog” or “homologue” as used herein in connection withherpesvirus proteins preferably means that a CMV gene of one CMVspecies, such as MCMV, can replace a homolog of said MCMV gene inanother CMV species, such as HCMV. (Schnee, M. et al., 2006, J Virol80:11658-11666). In other words homolog proteins preferably exhibit atleast one common protein function. Accordingly, it will be acknowledgedby a person skilled in the art that, for example, the homolog of UL50 ofHCMV is M50 of MCMV and vice versa. A person skilled in the art willacknowledge that two proteins which exhibit at least one common proteinfunction do not necessarily share the same amino acid sequence. Forexample, the gene product of m152 of MCMV and the gene product of US3 ofHCMV are also understood to be homologs as both retain MHC I moleculesin the ER.

Homologs or homologue proteins that display some degree of sequencehomology in MCMV and HCMV are listed in Rawlinson et al. (Rawlinson, W.D. et al., 1996, J Virol 70(12):8833-49). Further examples are the viralimmunevasins targeting the ligands for NK cell recpetors in human andmurine cells. In connection therewith it will be acknowledged that notall NKG2D ligands are homologs by sequence but by function. It will beacknowledged by a person skilled in the art that preferred human NKG2Dligands are MHC class-1-related protein A (MICA), MICB and UL16-bindingproteins (ULPB1-6). In connection therewith it is important to note thatULBP1 and ULBP2 also bind to mouse NKG2D (Sutherland, C. L. et al.,2006, Blood 108 (4): 1313-1319).

Alternatively and/or additionally, the term “homolog” or “homologue” asused herein in connection with herpesvirus proteins, preferably means apeptide, polypeptide or protein, wherein the gene encoding said peptide,polypeptide or protein is the gene of one herpesvirus species, such asHCMV, indicated to be a homolog of the gene of another species, such asanother herpesvirus species according to Fossum et al. (Fossum, E. etal. 2009, PLoS Pathog5(9):) or Davison et al. (Davison, A. J. et al.,2010, Vet Microbiol. 143(1):52-69 and Davison, A. J. et al., 2004,Compendium of Human Herpesvirus gene names, Reno).

Alternatively and/or additionally, the term “homolog” or “homologue” asused herein in connection with cellular proteins, such as a cellularligand as encoded by the beta-herpesvirus according to the presentinvention, preferably means that a protein and its homolog, i.e. itshomolog protein exhibit a common biological function. Preferably, twoproteins are homologs of each other if said two proteins show an aminoacid (aa) sequence identity and/or an identity of the nucleotidesequence of the genes encoding the proteins. More preferably saidsequence identity is of at least 50%, more preferably at least 75%, andmost preferably at least 80%, 90% or 95%. Nevertheless it is importantto note that as homolog proteins evolve, their 3D structure oftenremains more conserved than their sequence. Consequently, similaritiesin protein structure can be more reliable than sequence similarities forgrouping together distant homologs, which often retain some aspect ofsuch common biological function.

The term “receptor” as used herein preferably means a molecule expressedon the surface of a cell, whereby said molecule is capable of binding acellular ligand. A receptor-ligand binding as used herein is preferablycapable of initializing or inhibiting biochemical pathways and/or signalcascades when the proper ligand is binding to the receptor. It will beunderstood by a person skilled in the art that the cellular ligand(s)encoded by the beta-herpesvirus of the present invention is preferablyone which is, as such and when naturally occurring, expressed by a cellfrom a subject, such as a neighboring cell of the receptor expressingcell or a cell of the wider environment within an organism. In anembodiment of the beta-herpesvirus according to the present inventionthe gene encoding such cellular ligand is inserted into the genome ofthe beta-herpesvirus, the beta-herpesvirus is capable of expressing saidcellular ligand, preferably upon infection of a cell, irrespective ofwhether expression of said ligand is naturally occurring in saidinfected cell, i.e. without infection of the cell by saidbeta-herpesvirus. In a further embodiment the cellular ligand is acellular ligand which, when naturally occurring, is expressed uponinfection of the cell by the beta-herpesvirus according to the presentinvention expressing said ligand. In a still further embodiment thecellular ligand is a cellular ligand which, when naturally occurring, isexpressed in the absence of infection of the cell by thebeta-herepsevirus according to the present invention expressing saidligand. In connection therewith it is thus to be understood that abeta-herpesvirus of the present invention comprising a gene encoding acellular ligand may mediate expression of a cellular ligand which whennaturally occurring, is not expressed in the infected cell or is notexpressed upon infection of the cell infected by the betaherpesvirusaccording to the present invention.

The binding of a cellular ligand a with its respective receptorpreferably mediates a signal to the cell bearing said receptor toinitialize or inhibit a biochemical pathway and/or signal cascade. Thismay, for example, result in the cell's activation or de-activation, celldivision or cell death, or in certain molecules entering and/or exitingthe cell.

Receptors as used herein preferably are protein molecules, embedded ineither the plasma membrane of a cell in case of cell surface receptorsor the cytoplasm of a cell in case of nuclear receptors, to which one ormore specific types of signaling molecules, such as a cellular ligand asdisclosed herein, may bind.

A molecule which binds to a receptor is preferably called a ligand, andis, in an embodiment of the beta-herpesvirus according to the presentinvention, a peptide, a polypeptide, a protein or a small molecule, suchas a neurotransmitter, a hormone, a pharmaceutical drug, or a toxin.Each kind of receptor can bind only certain types or forms of ligands.Each cell typically has many receptors of many different types.

In an embodiment of the beta-herpesvirus of the present invention aligand, including a cellular ligand, is also understood to be asubstance that forms a complex with a biomolecule to serve a biologicalpurpose. In a narrower sense of said embodiment, a ligand is a signaltriggering molecule shich binds to a site on a target molecule which ispreferably a receptor for said ligand.

The binding of a ligand with a target molecule, such as a receptormolecule preferably occurs by intermolecular forces, such as ionicbonds, hydrogen bonds and van der Waals forces. The docking, i.e. theassociation, of ligand to target molecule, is usually reversible, i.e.dissociation of the ligand from the receptor may occur under specificreaction conditions. In an embodiment of the present inventionirreversible covalent binding between a ligand and its target molecule,such as the respective receptor, occurs.

The binding of a ligand to a receptor usually alters the conformation ofthe receptor particularly in case the receptor is a peptide, polypeptideor protein. The conformation of a receptor determines the function of areceptor. Ligand as referred to herein preferably comprise substrates,inhibitors, activators, and neurotransmitters.

Immune cells such as NK cells, express a variety of receptors that serveeither to activate or to suppress the immunologic activity of saidimmune cells, including the cytolytic activity thereof.

Natural killer cells, also referred to herein as NK cells, are a type ofcytotoxic lymphocyte that constitutes a major component of the innateimmune system. NK cells play a major role in the rejection of tumors,bacteria, parasites and cells infected by viruses. One particularimmunological activity mediated by NK cells is the release of smallcytoplasmic granules of proteins called perforin and granzyme that causethe target cell to die by apoptosis, i.e. programmed cell death and IFNγand TNFα release.

NK cells are defined as large granular lymphocytes, also referred toherein as LGL, and constitute the third type of cells differentiatedfrom the common lymphoid progenitor generating B and T lymphocytes. NKcells preferably do not express T-cell antigen receptors, also referredto herein as TCR, or Pan T marker CD3 or surface immunoglobulins (Ig) Bcell receptors. More preferably, NK cells preferably and herein expresssurface markers such as CD16 (FcγRIII) and CD56 in humans and NK1.1 orNK1.2 in C57BL/6 mice. Up to 80% of human NK cells also express CD8.

Given their strong cytolytic activity and the potential forauto-reactivity, the activity of NK cells is tightly regulated. NK cellsmust receive an activating signal which can come in a variety of formscomprising cytokines, Fc receptor, activating and inhibitory receptors.

As known to a person skilled in the art NK cells were found to notrequire activation in order to kill cells that are missing “self”markers of major histocompatibility complex, also referred to herein asMHC class I.

In order for NK cells to defend the body against viruses and otherpathogens, NK cells require mechanisms that enable the determination ofwhether a cell is infected or not. The exact mechanism has not yet beenfinally elucidated, but recognition of an “altered self” state isthought to be involved. To control their cytotoxic activity, NK cellspossess two types of surface receptors: activating receptors andinhibitory receptors. Most of these receptors are not unique to NK cellsand can be present in some T cell subsets as well.

The relative balance of signals from these receptors regulates NK cellactivity (Lanier, L. L. et al., 2001, Nature Immunol 2:23-27; Moretta,A. et al., 2001, Annu Rev Immunol 19:197-223; Ravetch, J. V. and Lanier,L. L. 2000, Science 290:84-89 and Lopez-Botet, M. et al., 2000, HumImmunol 61:7-17).

NK cell receptors, also referred to herein as NKR, can be subdividedinto activating or inhibitory receptors. Although the extracellulardomains of the various NKRs are extremely diverse, their intracellulardomains are mostly conserved so that inhibitory or activating receptorsshare common inhibitory or activating signaling pathways. Inhibitoryreceptors contain a tyrosine-based inhibitory motif (ITIM) in theirintracellular domain. Receptor ligation triggers tyrosinephosphorylation by a Src family kinase. This recruits SHIP-1 to themembrane, which then degrades phosphatidylinositol-3,4,5-trisphosphateto phosphatidylinositol-3,4-bisphosphate; SHP-1 or SHP-2 can also berecruited to the membrane at this time.

Activating NK cell receptors, such as NKG2D, lack ITIMs, but posses apositively charged arginine residue in their transmembrane domain whichallows them to interact with adaptor proteins such as DAP10, DAP12,FcεRI-γ or CD3-ξ (Lanier, L. L. 2008, Nat Immunol 9(5):495-502.). Theseadaptors bear tyrosine-based activation motifs (ITAMs), which arephosphorylated upon receptor engagement, also by a Src family kinase.Syk, ZAP-70, and PI₃K or Grb2 are then recruited to the membrane, wherethey mediate actin cytoskeleton reorganization, cell polarization,release of cytolytic granules, and the transcription of many cytokineand chemokine genes. The engagement of NKG2D leads to NK cellcytotoxicity and cytokine secretion or to a co-stimulation of CD8+ Tcells.

In connection therewith a person skilled in the art will acknowledgethat the maturation of NK cells is characterized by a high frequency ofNK cells expressing the most mature phenotype CD27^(low)CD11b^(high).For example a high frequency of NK cells expressing the most maturephenotype CD27^(low)CD11b^(high) is observed in WT-MCMV infected mice.Additionally and alternatively, NK-cell expression of IFN-γ and CD69,but also several other markers, can be used to assess the activation ofNK cells. In connection therewith a person skilled in the art knowsassays for assassing wheter NK cell is activated or not. For example, NKcells can be analysed by flow cytometry after surface staining withanti-CD69 (H1.2F3) and for the detection of IFN-γ expression by NKcells, incubation in medium supplemented with 10% of FCS (Gibco), IL-2and Brefeldin A (eBioscience) is needed.

Natural killer cells express inhibitory receptors specific forpolymorphic MHC molecules, which enables them to mediate “missingself-recognition”, which provides the capacity to attack cells of thebody that extinguish expression of MHC class I molecules. Theseinhibitory receptors recognize MHC class I alleles, which is regarded asan explanation why NK cells kill cells possessing low levels of MHCclass I molecules. This inhibition is crucial to the role played by NKcells. MHC class I molecules mediate a main mechanism by which cellsdisplay viral or tumor antigens to cytotoxic T-cells. A commonevolutionary adaption to this is seen in both intracellular microbes andtumours is a chronic down-regulation of these MHC I molecules, renderingthe cell impervious to T-cell mediated immunity. It is believed that NKcells, in turn, evolved as an evolutionary response to this adaption, asthe loss of the MHC would deprive these cells of the inhibitory effectof MHC and would render these cells vulnerable to NK cell mediatedapoptosis.

More specifically, inhibitory NK cell receptors comprise an ITIM motifin the intracellular domain of said NK cell receptors. Ligation ofinhibitory receptors via their ligand, such as MHC-I, leads tophosphorylation of motifs and initiates the signaling cascade involvingtyrosine phosphatase such as SHP-1 and SHP-2 and results indephosphorilation of activating signaling motif and NK cell inhibiton.(Lanier, L. L. 2003, Curr Opin Immunol 15(3):308-14). The inhibitory NKcell receptors include killer immunoglobulin-like receptors (KIR) andimmunoglobulin-like receptors (LIR) in humans and the Ly49 family ofreceptors in the mouse, as well as CD94-NKG2A receptor in humans andmice (Vilches, C. and Parham, P. 2002, Annu Rev Immunol 20:217-51;Moretta, L. A. 2004, Embo J 23(2):255-259). Ligands for the inhibitoryKIR receptor family, such as LIR and Ly49 receptors are classical MHC-Imolecules, while CD94-NKG2A receptor binds to non-classical MHC-Imolecule, HLA-E in humans and Qa-1 in mice (Raulet, D. H. 2003, Nat RevImmunol 3(10):781-90). Recognition of MHC-I molecules is also requiredduring the development of NK cells and for reaching their functionalcompetence (reviewed in: Elliott, J. M. and Yokoyama, W. M. 2011, TrendsImmunol 32(8):364-72; Vivier, E. et al., 2011, Science 7;331(6013):44-49).

Furthermore, the family of natural cytotoxicity receptors, also referredto herein as NCR receptors, which comprises NKp30, NKp44, NKp80 andNKp46, is almost exclusively expressed by NK cells. All four receptorshave been extensively studied in human NK cells, whereas NKp46 has beencharacterized in mice as well. In contrast to NKp30, NKp80 and NKp46,only NKp44 is not expressed on all NK cells, but is only found onactivated NK cells. NCRs ligands are diverse and include viralhaemagglutinins (NKp46 and NKp44), heparan sulphate proteoglycans (NKp30and NKp46) and the nuclear factor HLA-B-associated transcript 3 (BAT3)(NKp30) 47 and the activation-induced C-type lectin (AICL) (NKp80).However, no endogenous cellular NCR ligands have been identified as yet.(Raulet, D. H. and Guerra, N. 2009, Nat Rev Immunol. 9(8):568-80)

Preferably, NK cells as used herein are different from Natural Killer Tcells.

Furthermore, NK cell receptor types, both with inhibitory as well asactivating function, can be differentiated by structure.

Based on their chemical structure NK cell receptors are classified intotwo families:

-   -   a) receptors containing Ig-like ectodomains and    -   b) receptors containing C-type lectin-like domains        (Raulet, D. H. 2003, supra).        The latter group include Ly49, NKG2 and NKRp1 receptors.

The following is a compilation of the most important NK cell receptortypes. CD94 also referred to as NKG2, which are C-type lectin familyreceptors, comprising NKG2D, are conserved in both rodents and primatesand identifies non-classical (also non-polymorphic) MHC I molecules likeHLA-E. Expression of HLA-E at the cell surface is dependent on thepresence of a nonamer peptide epitope derived from the signal sequenceof classical MHC class I molecules, which is generated by the sequentialaction of signal peptide peptidase and the proteasome.

Ly49 represents C-type lectin family receptors which are of multigenicpresence in mice, while humans have only one pseudogenic Ly49. Ly49 isthe receptor for classical (polymorphic) MHC I molecules. It will beimmediately understood by a person skilled in the art that multigenic asused herein preferably refers to a receptor which is encoded by multiplegenes. For example, C-type lectin family receptors are encoded bymultiple genes in the NK gene complex (NKC) on mouse chromosome 6.

More particularly, Ly49 receptors are type II transmembrane proteinsencoded by polymorphic and polygenic gene complex on chromosome 6(reviewed in Ravetch, J. V. and Lanier, L. L., 2000, Science,290(5489):84-9.). The number of Ly49 genes varies among different mousestrains. Although many inhibitory receptors are shared among differenthaplotypes (e.g. Ly49A, Ly49C, Ly49G2, and Ly49I), their contribution ininhibitory signalling is different with regards to MHC-Ihaplotype-restricted education imposed during NK cell development. Thenumber of activating genes varies a great deal among strains, from one(Ly491) in BALB/c to seven (Ly49d, u, p3, p1, w, m, h) in NOD mice(Carlyle, J. R. et al., 2008, Semin Immunol 20(6):321-30).

KIR, also referred to herein as Killer-cell immunoglobulin-likereceptors, belong to a multigene family of more recently-evolved Ig-likeextracellular domain receptors. KIR are present in non-human primatesand are the main receptors for both classical MHC I (HLA-A, HLA-B,HLA-C) and also non-classical HLA-G in primates. Some KIRs are specificfor certain HLA subtypes.

ILT or LIR, also referred to herein as leukocyte inhibitory receptors,are recently-discovered members of the Ig receptor family.

NKG2D is an activating receptor for the NKG2 receptor family expressedon NK cells, NKT cells, γδ T cells and CD8⁺ T cells. NKG2D recognizescell surface molecules structurally related to MHC class I proteinsinduced by infection or any other type of cellular stress. Theinvolvement of NKG2D leads to NK cell cytotoxicity and cytokinesecretion or to a co-stimulation of CD8⁺ T cells.

NKG2D is a type-2 trans-membrane glycoprotein expressed as a disulfidelinked homodimer on the cell surface (Diefenbach, A. et al., 2002, NatImmunol, 3:1142-9; Jamieson, A. M. et al., 2002, Immunity, 17:19-29). Itis encoded by the KLRK1 (killer cell lecitin-like receptor subfamilymember 1) gene located on a mouse chromosome 6 and in the syntenicposition on human chromosome 12 (Houchins, J. P. et al., 1991, J ExpMed, 173:1017-20). NKG2D has no signaling motif and therefore associateswith signal-transducing proteins through charged residues in thetrans-membrane region. In mice, alternative RNA splicing results in twoNKG2D isoforms, the long (NKG2D-L) and short (NKG2D-S) isoform whichdiffer in 13 amino acids (Diefenbach, A. et al., 2002, supra; Gilfillan,S. et al., 2002, Nat Immunol, 3:1150-5). The NKG2D-L isoform pairs withthe DAP10 signaling molecule, while NKG2D-S associates either with DAP10or DAP12. Resting mouse NK cells express very little NKG2D-S, but theexpression is induced after NK cell activation. Neither isoform can bedetected in resting CD8⁺ T cells but after T-cell receptor (TCR)stimulation the expression of both isoforms is upregulated. Because CD8⁺T cells do not express DAP12, the two NKG2D isoforms that are expressedby activated T cells can interact only with DAP10, whereas activated NKcells can transmit signals through DAP10 and DAP12. In humans there isonly one isoform which corresponds to the long form in mouse and it onlyinteracts with DAP10 (Bauer, S. et al., 1999, Science, 285:727-9; Wu, J.et al., 1999, Science, 285:730-2; Rosen, D. B. et al., 2004, J Immunol,173:2470-8). NKG2D is expressed by all NK cells, most NKT cells, asubset of γδ T cells, all human CD8⁺ T cells, activated mouse CD8⁺ Tcells and a subset of CD4 T cells (Diefenbach, A. et al., 2002, supra;Jamieson, A. M. et al., 2002, Immunity, 17:19-29; Bauer, S. et al.,1999, supra; Diefenbach, A. et al., 2000, Nat Immunol, 1:119-26;Girardi, M. et al., 2001, Science, 294:605-9; Ehrlich, L. I. et al.,2005, J Immunol, 174:1922-31). On NK cells NKG2D serves as a primaryactivating receptor meaning that the engagement of NKG2D can overrideinhibitory signals (Cerwenka, A. et al., 2001, Proc Natl Acad Sci USA,98:11521-6). NKG2D receptor on CD8⁺ T cells acts as a co-stimulatoryreceptor which augments TCR-induced responses (Groh, V. et al., 2001,Nat Immunol, 2:255-60; Maasho, K. et al., 2005, J Immunol, 174:4480-4;Markiewicz, M. A. et al., 2005, J Immunol, 175:2825-33).

Human NKG2D was originally identified in 1991 as an orphan receptor thatis expressed on NK cells (Houchins, J. et al., 1991, J. Exp. Med.173(4):1017-20). The mouse (Vance, R. et al., 1997, Eur. J Immunol27(12):3236-41; Ho, E. et al., 1998, Proc Natl Acad Sci USA95(11):6320-5), rat (Berg, S. F. et al., 1998, Int. Immunol.10(4):379-85), and porcine (Yim, D. et al., 2001, Immunogenetics53(3):243-9). Homologs of NKG2D have also been identified. Interspeciesamino acid (aa) sequence identities range from 52-78% for the entireprotein, whereby mouse and rat are the most closely related sequences,and from 72-90% within the lectin domain of NKG2D. Its function wasfirst described in 1999 by two separate groups investigating MICA/MICBligands (Bauer, S. et al., 1999, supra) or signal transduction throughthe DAP10 adapter protein (Wu, J. et al., 1999, supra). More recently,several additional ligands have also been reported (Cerwenka, A. et al.,2000, Immunity 12(6):721-7, Diefenbach, A., et al. 2000, supra; Cosman,D. et al., 2001, Immunity. 14(2):123-33).

NKG2D ligands are distantly related homologs of the MHC I proteins andare characterized by a striking structural diversity, differentexpression patterns and regulation mechanisms.

Preferred human NKG2D ligands are MHC class-1-related protein A, alsoreferred to herein as MICA, MHC class-1-related protein B, also referredto herein as MICB, and UL16-binding proteins 1 to 6, also referred toherein as ULPB1 to 6. MICA (ENSG00000204520 according to the ENSEMBLdatabase) and MICB (ENSG00000231372 according to the ENSEMBL database)),encoded by the genes within human MHC (Bauer, S. et al., 1999, supra;Groh, V. et al., 1996, Proc Natl Acad Sci USA 93:12445-50) are the onlyNKG2D ligands containing three immunoglobulin-like domains (α1, α2 andα3), but unlike MHC molecules, they do neither associate with β2microglobulin nor do they bind antigenic peptides. All other NKG2Dligands are related to MHC I molecules but contain only α1 and α2domains. In an embodiment of the beta-herpesvirus according to thepresent invention wherein the cellular ligand comprises at least oneimmunoglobulin-like domain, the cellular ligand comprises an α1 domainand an α2 domain. In a further embodiment of the beta-herpesvirusaccording to the present invention the cellular ligand comprises an α1domain, an α2 domain and an α3 domain, wherein the cellular ligand ispreferably selected from the group comprising MICA and MICB.

Although named by their ability to bind HCMV protein UL16, only thefirst two identified ULBP proteins ULBP1 (ENSG00000111981 according tothe ENSEMBL database)), ULBP2 (ENSG00000131015 according to the ENSEMBLdatabase)) and the subsequently described ULBP5 bind this viral protein(Cosman, D. et al., 2001, Immunity, 14:123-33; Radosavljevic, M. et al.,2002 Genomics, 79:114-23; Bacon L. et al., 2004, J Immunol,173:1078-84). Like MIC proteins, ULBP5 and ULBP6 are trans-membraneproteins, while proteins ULBP1, ULBP2 and ULBP3 (ENSG00000131019according to the ENSEMBL database)) are anchored to the membrane viaglycosylphosphatidylinositol (GPI) (Eleme, K. et al., 2004, J Exp Med,199:1005-10). The ULBP family is also known as the retinoic acid earlytranscript 1 (RAET-1) family since they show sequence homology to themouse retinoic acid early 1 (RAE-1) proteins (Nomura, M. et al., 1994,Differentiation, 57:39-50; Cerwenka, A. et al., 2000, Immunity,12:721-7; Diefenbach, A. et al., 2001, Nature, 413:165-71). Accordingly,in human and in case of the beta-herpesvirus according to the presentinvention for us in the vaccination of a human ligands for NKG2D arepreferably selected from the group comprising MHC class-1-relatedproteins and UL16-binding proteins. MHC class-1-related proteins arepreferably selected from the group comprising MICA and MICB.UL16-binding proteins are preferably selected from the group comprisingULPB1, ULPB2, ULPB3, ULPB4, ULPB5 and ULPB6. With regard to mouse NKG2Dligands there is a first family of ligands which is referred to as theRAE-1 family. Said RAE-1 family comprises RAE-1α, RAE-1β, RAE-1γ, RAE1-δand RAE-1ε, which are highly related to each other (>85% amino acidhomology) and differentially expressed in various mouse strains. Asecond family of mouse NKG2D ligands is the H60 family that comprisesthree members of the H60 family, namely H60a, H60b and H60c, of whichH60a is a minor histocompatibility antigen (Malarkannan, S. et al.,1998, JI Immunol, 161:3501-9; Takada, A. et al., 2008, J Immunol,180:1678-85; Whang, M. I. et al., 2009, J Immunol, 182:4557-64).Finally, murine UL16 protein-like transcript 1, also referred to hereinas MULT-1, is the sole member of the third family of mouse NKG2D ligands(Carayannopoulos, L. N. et al., 2002, J Immunol, 169:4079-83;Diefenbach, A. et al., 2003, Eur J Immunol, 33:381-91). Accordingly, inmice ligands for NKG2D are preferably selected from the group comprisingproteins of the RAE-1 family, proteins of the H60 family and murine UL16protein-like transcripts. Proteins of the RAE-1 family are preferablyselected from the group comprising RAE-1α, RAE-1β, RAE-1γ, RAE1-δ andRAE-1ε. Proteins of the H60 family are preferably selected from thegroup comprising H60a, H60b and H60c. Murine UL16 protein-liketranscripts are preferably selected from the group comprising MULT-1.

In an embodiment of the beta-herpesvirus according to the presentinvention the cellular ligand is a trans-membrane protein. In apreferred embodiment wherein the cellular ligand is a trans-membraneprotein the cellular ligand is preferably selected from the groupcomprising MIC proteins, ULBP5 and ULBP6. In a further embodiment thecellular ligand is a MIC protein preferably selected from the groupcomprising MICA and MICB

A trans-membrane protein as used herein preferably means a protein whichis extending through the membrane of a cell. More preferably, atrans-membrane protein as used herein is an integral membrane proteinwhich extends from one side of a membrane of a cell to the other side ofthe membrane. Furthermore, a trans-membrane protein as used hereinpreferably is a polytopic protein that spans an entire biologicalmembrane.

In an embodiment of the beta-herpesvirus according to the presentinvention the cellular ligand is a protein anchored to or in themembrane via glycosylphosphatidylinositol, also referred to herein asGPI. In an embodiment the cellular ligand anchored to or in the membranevia GPI is preferably selected from the group comprising ULBP1, ULBP2and ULBP3.

A “glycosylphosphatidylinosityl” which is also referred to herein as“GPI-anchor”, is preferably a glycolipid which preferably is attached toa terminus, preferable the C-terminus, of a protein duringposttranslational modification. The GPI-anchor is preferably composed ofa phosphatidylinositol group linked through a carbohydrate-containinglinker, i.e. glucosamine and mannose glycosidically bound to theinositol residue, and via an ethanolamine phosphate (EtNP) bridge to theterminal amino acid of a protein. Preferabyl, the terminal amino acid isthe C-termunal amino acid. The two fatty acids within the hydrophobicphosphatidyl-inositol group anchor the protein to or in the membrane ofa cell.

A protein anchored to or in the membrane viaglycosylphosphatidylinositol also referred to herein as a GPI-linkedprotein, is preferably a protein which contains a signal peptidedirecting the protein into the endoplasmic reticulum (ER). The terminusof the protein, such as the C-terminus, is preferably composed ofhydrophobic amino acids that stay inserted in the membrane of the ER.Such hydrophobic end is then cleaved off by an enzyme and replaced bythe GPI-anchor. As the protein processes through the secretory pathwayof the ER, it preferably is transferred via vesicles to the Golgiapparatus and finally to the extracellular space where it remainsattached to the exterior leaflet of the cell membrane. In a preferredembodiment the GPI anchor is the sole means of attachment of suchproteins to the membrane. It will be understood by a person skilled inthe art that cleavage of the GPI-anchor by phospholipases will result incontrolled release of the protein from the membrane.

It is the merit of the present inventor to have found that the insertionof at least one gene encoding a cellular ligand, such as NKG2D, into thegenome of a beta-herpesvirus, results in expression of said cellularligand which overrides inhibitory signals delivered by self-MHC class Iproteins and thus triggers activation of an immune cell, moreparticularly NK cells.

The importance of signaling pathways in beta-herpesvirus control inducedby receptor-ligand interaction, such as NKG2D signaling pathway, is bestillustrated by the sophisticated mechanism that HCMV and MCMV havedeveloped to avoid NKG2D-mediated immune control.

It is acknowledged by a person skilled in the art that more than half ofthe CMV genes encode gene products interfering with different immunemechanisms at all stages of the immune system of an organsim infectedwith CMV. Such gene products which interfere with different immunemechanisms are referred to herein as so-called immunoevasins. The geneenconding an immunevasin is also referred to herein as immunevasive geneor immune modulatory gene.

Infection of the respective host organism with either of HCMV and MCMVup-regulates the transcription of NKG2D ligands in said host organisms,which can result in NKG2D-mediated lysis by NK cells of cells infectedby HCMV and MCMV, respectively. Both viruses have developed differentevasive mechanisms using immunevasins to prevent and/or at leastcounteract the expression of NKG2D ligands on the cell surface. Suchimmunevasins which downregulate the expression of NKG2D ligands on thecell surface of infected cells are also referred to herein as geneproducts regulating NK cell response.

More particularly, HCMV protein UL16 which is an immune modulatory geneencoding a gene product regulating NK cell response, binds NKG2D ligandsMICB, ULBP1, ULBP2 and ULBP6 in the endoplasmatic reticulum (ER) andredirects these ligands to the lysosomes for sequestration (Cosman, D.et al., 2001, supra; Welte, S. A. et al., 2003, Eur J Immunol,33:194-203; Dunn, C. et al., 2003, J Exp Med, 197:1427-39; Wu, J. etal., 2003, J Immunol, 170:4196-200; Vales-Gomez, M. et al., 2003, BMCImmunol, 4:4; Rolle, A. et al., 2003, J Immunol, 171:902-8). The abilityof UL16 to bind its ligands depends critically on the presence of aglutamine (MICB) or closely related glutamate (ULBP1 and ULBP2) atposition 169. An arginine residue at this position however, as found forexample in MICA or ULBP3, would cause steric clashes with UL16 residues.The inability of UL16 to bind MICA and ULBP3 can therefore be attributedto single substitutions at key NKG2D ligand locations (Muller, S. etal., 2010, PLoS Pathog, 6(1):e1000723). UL16 doesn't bind ULBP3-5. Thestructure of UL16 revealed that this viral protein mimics a centralbinding motif of otherwise structurally unrelated NKG2D and thusenabling the virus to evade several diverse NKG2D ligands (Muller, S. etal., 2010, supra). The gene product of another HCMV immunomodulatorygene, namely the gene product of UL142, retains newly synthesizedfull-length MICA in the cis-Golgi compartment (Ashiru, O. et al., 2009,J Virol, 83:12345-54; Chalupny, N. J. et al., 2006, Biochemical andbiophysical research communications, 346(1):175-81). Interestingly, theMICA*008 allele, which lacks the cytoplasmatic domain, is resistant tothe action of UL142. Since MICA*008 is frequently found in humans, thissuggests that HCMV exerts during co-evolution selective pressure to thehost which drives diversity and polymorphism of NKG2D ligands. Inaddition to targeting already synthesized proteins, HCMV employs miRNAsto prevent the translation of ligands for NKG2D. HCMV encodesmiRNA-UL112 that competes with endogenous miRNA for binding to the3′-UTR (untranslated region) of the MICA transcript thus repressing thetranslation of this NKG2D ligand (Stern-Ginossar, N. et al., 2007,Science, 317:376-81; Stern-Ginossar, N. et al., 2008, Nat Immun,9:1065-73; Wilkinson et al., 2008, J Clin Virol, 41(3):206-12). In anembodiment of the beta-herpesvirus of the present invention thebeta-herpesvirus herpesvirus comprises the deletion of at least onemiRNA. In a further embodiment the miRNA is capable of binding atranscript of the cellular ligand. In a further embodiment the miRNA iscapable of repressing the translation of the gene coding for thecellular ligand. In a still further embodiment the miRNA is miRNA-UL112,also referred to herein as hcmv-miR-UL112 (MIMAT0001577AAGUGACGGUGAGAUCCAGGCU (MirBase) (Stern-Ginossar et al., 2008, supra;Stern-Ginossar et al., 2007, supra)

In an embodiment of the beta-herpesvirus of the present invention,wherein binding of a gene product regulating NK cell response is capableof binding the cellular, the binding of the gene product regulating NKcell response and the cellular ligand results in reduction of expressionof the cellular ligand. In connection therewith it will be acknowledgedthat a person skilled in the art will know tests for assessing wheterthe expression of the cellular ligand is reduced. Such test is, forexample, described in Cosman et al. (Dunn, C. et al., 2003, J Exp Med,197(11):1427-39). More particularly, FACS analysis of surface expressionof cellular ligands and assays for determining NK cell cytotoxicity canbe performed.

MCMV also encodes immunoevasins that prevent the accessibility of mouseNKG2D ligands to the cell surface (Lenac, T. et al., 2008, Med MicrobiolImmunol, 197:159-66). The product of the m152 gene, initially describedas a negative regulator of MHC-I molecules (Ziegler, H. et al., 1997,Immunity, 6:57-66), retains the RAE-1 family of proteins in theERGIC/cis-Golgi compartment (Krmpotic, A. et al., 2002, Nat Immunol,3:529-35; Lodoen, M. et al., 2003, J Exp Med, 197:1245-53; Arapovic, J.et al., 2009, J Virol, 83:8198-207). Not all RAE-1 isoforms, however,are equally susceptible to MCMV regulation. RAE-1δ is more resistant toMCMV than other RAE-1 isoforms which is, at least in part, due to theabsence of the PLWY motiv in the RAE-1δ cytoplasmatic domain (Arapovic,J. et al., 2009, supra). The product of the m155 gene causes thedegradation of the H60 protein, although the underlying molecularmechanism is not yet entirely understood. The product of the m145 genebinds MULT-1 after it leaves the ER and makes MULT-1 more susceptiblefor m138-mediated degradation (Krmpotic, A. et al., 2005 J Exp Med,201:211-20). Finally, the product of m138, also known as fcr-1, assistsin diminishing the expression of MULT-1, H60 and RAE-1ε by interferingwith their recycling on the cell surface and redirecting them tolysosomes for degradation (Lenac, T., 2006, J Exp Med, 203:1843-50;Wilkinson, G. W. et al., 2008, J Clin Virol, 41(3):206-12.)).

As has been outlined above receptors, such as NKG2D, may serve asprimary activating receptors, particularly on NK cells. In a preferredembodiment of the beta-herpesvirus of the present invention thus atleast one gene encoding a cellular ligand, preferably a gene encoding atleast one ligand for NKG2D, is introduced into the genome of thebeta-herpesvirus. Expression of said cellular ligand is preferablymediated and occurring, respectively, upon infection of a cell with saidbeta-herpesvirus whereby the cell is preferably permissive for infectionwith the beta-herpesvirus.

In a further preferred embodiment of the beta-herpesvirus of the presentinvention at least one immune modulatory gene is deleted from the genomeof the beta-herpesvirus. It is also within the present invention thatmore than one immune modulatory gene is deleted from the genome of thebeta-herpesvirus. In CMV simultaneous deletion of several immunemodulatory genes as well as other genes encoding gene products withunknown function or functions other than immunevasion has been described(Cicin-Sain et al., 2007, supra). In a further embodiment of thebeta-herpesvirus of the present invention the additional deletion of atleast one non-essential gene coding for a gene product with a functionother than immunevasion or immunemodulation is also considered. The termnon-essential gene as used herein preferably means a gene which encodesa gene product, wherein a beta-herpesvirus deficient in said geneproduct is not attenuated in vitro. An essential gene as used hereinpreferably means a gene which encodes a gene product, wherein abeta-herpesvirus deficient in said gene product is not capable ofreplicating in vitro. Additionally and alternatively, The an essentialgene as used herein in connection with herpesvirus proteins, preferablymeans a gene, indicated to be an essential gene according to Liu et al.(Liu et al., PNAS 2003). In an embodiment of the present invention anessential gene of HCMV is preferably selected from the group comprisingUL32, UL34, UL37.1, UL44, UL46, UL48, UL48, UL49, UL50, UL51, UL52,UL53, UL54, UL55, UL56, UL57, UL60, UL70, UL71, UL73, UL75, UL76, UL77,UL79, UL80, UL84, UL85, UL86, UL87, UL89.1, UL90, UL91, UL92, UL93,UL94, UL95, UL96, UL98, UL99, UL100, UL102, UL104, UL105, UL115 andUL122.

As far as the deletion of at least one immune modulatory gene iscontemplated herein said at least one immune modulatory gene ispreferably selected from the group comprising genes encoding geneproducts affecting antigen presentation, cytokine response, thecomplement system and humoral immunity. More preferably, the deleted atleast one immune modulatory gene is preferably selected from the groupcomprising a gene encoding a gene product that down-regulates MHC I toavoid CTL response; a gene enconding a gene product that evades the NKcell response, such as a gene enconding gene product which prevents theaccessibility of mouse NKG2D ligand to the cell surface; a geneenconding a gene product that interferes with MHC II presentation; agene enconding a gene product that down-regulates adhesion molecules; agene enconding a gene product that interacts with IL-1; a gene encondinga gene product that activates TGF-β.

In an embodiment of the beta-herpesvirus of the present invention wheremore than one immune modulatory gene is deleted from the genome of thebeta-herpesvirus, only immune modulatory genes from one particular groupof immune modulatory genes are deleted, e.g. only immune modulatorygenes are deleted the gene product of which results in preventing theaccessibility of an NKG2D ligand to the cell surface, more preferably ofone particular NKG2D ligand. In a further embodiment immune modulatorygenes from two, three, four, five or more of the above mentioned immunemodulatory genes are deleted. In a still further embodiment each and anyimmune modulatory gene are deleted in combination with each and anyother immune modulatory gene.

In an embodiment of the beta-herpesvirus according to the presentinvention a gene product encoded by an immune modulatory gene ispreferably a gene product regulating NK cell response and morepreferably is encoded by an immune modulatory gene preferably selectedfrom the group comprising UL16, UL18, UL40, UL 141 (Prod'homme V et al.,J Gen Virol. 2010; Tomasec P et al., Nat Immunol. 2005), UL142, m152,m155, m145 and m138. In an embodiment wherein the beta-herpesvirus is anHCMV a gene product encoded by an immune modulatory gene is a geneproduct regulating NK cell response and more preferably is encoded by animmune modulatory gene preferably selected from the group comprisingUL16, UL18, UL40, UL142. In an embodiment wherein the beta-herpesvirusis an MCMV a gene product encoded by an immune modulatory gene is a geneproduct regulating NK cell response and more preferably is encoded by animmune modulatory gene preferably selected from the group comprisingm152, m155, m145 and m138.

In an embodiment of the beta-herpesvirus according to the presentinvention wherein the gene product is regulating MHC class Ipresentation the immune modulatory gene preferably selected from thegroup comprising US6, US3, US2, UL18, US11, UL83 and UL40.

It will be acknowledged that a person skilled in the art is aware offurther examples of an immune modulatory gene of each and any of theabove mentioned groups of immune modulatory genes as well as of othergroups of immunevasive genes regulating the immune response of theinfected host to the advantage of viral replication, viral spread and/orviral infection. The deletion of each and any of these immune modulatorygenes from the beta-herpesvirus according to the present invention isencompassed by the present invention and is preferably advantageous forthe vaccine and/or vector properties as well as for the immune responsemediated by said vaccine and/or vector of the present invention.Furthermore, it will be immediately understood that deletion of one ormore than one immune modulatory gene which encodes one or more than oneimmunevasin(s) regulating the immune response of the infected host tothe advantage of the virus preferably results in attenuation of thebeta-herpesvirus of the present invention.

In a most preferred embodiment of the beta-herpesvirus according to thepresent invention at least one immune modulatory gene is deleted whichencodes an immunevasin which regulates NK cell response and even morepreferably is capable of binding the cellular ligand comprised in thebeta-herpesvirus of the present invention. It is also preferred that ifmore than one such cellular ligand is comprised in the beta-herpesvirusof the present invention for at least one, preferably each and any ofsuch cellular ligand at least one immune modulatory gene is deletedwhich encodes an immunevasin which regulates NK cell response and whichis capable of binding said cellular ligand comprised in thebeta-herpesvirus of the present invention. In connection therewith ithas to be noted that dependent on the particular beta-herpesvirus andthe particular cellular ligand which is comprised in thebeta-herpesvirus of the present invention such deletion of animmunevasin which regulates NK cell response and which is capable ofbinding said cellular ligand comprised in the beta-herpesvirus isadvantageous. It is important to understand that in some embodiments ofthe beta-herpesvirus of the present invention such deletion isnevertheless not necessary as overexpression of the cellular ligand mayoverride the inhibitory capacity of the respective immunevasin whichregulates NK cell response and which is capable of binding said cellularligand comprised in the beta-herpesvirus. Nevertheless, it will beimmediately understood that, although not necessary, in some embodimentsit is preferred to delete the inhibitory gene which inhibits a or theparticular interaction of the particular receptor ligand, e.g. NK cellreceptor ligand, expressed by the beta-herpesvirus according to thepresent invention.

In an embodiment of the beta-herpesvirus of the present invention,wherein the beta-herpesvirus of the present invention encodes at leastone additional heterologous nucleic acid and wherein the at least oneadditional heterologous nucleic acid is a functional nucleic acid, thefunctional nucleic acid is preferably selected from the group comprisingantisense molecules, ribozymes and RNA interference mediating nucleicacids. It is a further embodiment that the at least one additionalheterologous nucleic acid is a heterologous nucleic acid coding for apeptide, oligopeptide, polypeptide or protein wherein preferably thepeptide, oligopeptide, polypeptide or protein constitutes or comprisesat least one antigen. In a further preferred embodiment an immunemodulatory gene such as US11 is used as insertion site for the at leastone additional heterologous nucleic acid. In an embodiment the immunemodulatory gene, such as US11 is deleted by insertion of the at leastone additional heterologous nucleic acid, which preferably codes for apeptide, oligopeptide, polypeptide or protein wherein preferably thepeptide, oligopeptide, polypeptide or protein constitutes or comprisesat least one antigen. In a further preferred embodiment an immunemodulatory gene is deleted by insertion of a gene into thebeta-herpesvirus genome comprising the at least one additionalheterologous nucleic acid at the position of the immune modulatory gene.For example US11 encodes an immunoevasin and its deletion will (i) leadto attenuation of the vaccine strain and (ii) will improve antigenpresentation in the HLA class I pathway. Nevertheless it will beimmediately understood that other ORFs encoding HCMV immunoevasins canbe used as insertion sites. Such sites of insertion are preferablyselected form the group comprising immune modulatory genes, wherein theimmune modulatory gene preferably is selected from the group comprisingUL14, UL18, UL141, UL142, US2 and US6. In a further preferred embodimentthe at least one additional heterologous nucleic acid is inserted intothe beta-herpesvirus genome into a sequence coding for an immunodominantCMV T cell epitope, for example IE1 or pp 65. In a further preferredembodiment the sequence coding for an immunodominant CMV T cell epitopeis replaced with a sequence of an epitope of an antigen as disclosedherein.

In connection therewith it is important to understand that certainbeta-herpesvirus strains may not comprise all immune modulatory genes.For example, the BAC cloned genome of HCMV strain TB40E (Sinzger et al.,2008, supra) already lacks US2 and US6. In connection therewith it willalso be understood by a person skilled in the art that the authenticand/or endogenous promotor of the immune modulatory gene is preferablyused for expression of the at least one additional heterologous nucleicacid. For example, the authentic US11 promoter is preferably used fordriving the expression of the influenza HA. A person skilled in the artwill know other promoters, such as the cellular PGK promoter, promoterof the cellular phosphoglycerate kinase (PGK) housekeeping gene, theviral MCMV MIEP or the HCMV MIEP which are equally useful. It isimportant to note that in case one of the viral MCMV MIEP or the HCMVMIEP is used, a person skilled in the art will know that depending onthe particular beta-herpesvirus strain used, the insertion preferablyoccurs in opposite direction to the already present copy of theseelements, for example in the TB40E-ULBP2 genome.

In certain embodiments of the beta-herpesvirus according to the presentinvention the beta-herpesvirus of the present invention is deficient inone or more additional gene product(s) each encoded by an additionalimmune modulatory gene other than the immune modulatory gene whichencodes an immunevasin which preferably regulates NK cell response andwhich is capable of binding said cellular ligand comprised in thebeta-herpesvirus. In a further embodiment no immune modulatory genewhich encodes an immunevasin which regulates NK cell response and whichis capable of binding said cellular ligand comprised in thebeta-herpesvirus is deleted but an immunevasive gene is deleted which isor is not regulating NK cell response and which is not capable ofbinding said cellular ligand comprised in the beta-herpesvirus.

Furthermore, in an embodiment of the beta-herpesvirus according to thepresent invention wherein the beta-herpesvirus of the present inventioncomprises a deletion of the coding sequence of the additional immunemodulatory gene the coding sequence of more than one immune modulatorygene is preferably deleted. Accordingly, the beta-herpesvirus isdeficient in one or more additional gene product(s) each encoded by anadditional immune modulatory gene.

In an embodiment of the beta-herpesvirus of the present invention thebeta-herpesvirus comprises the deletion of at least one miRNA, whereinsaid miRNA is preferably an miRNA which is capable of binding atranscript of the cellular ligand encoded by the beta-herpesvirus of thepresent invention. Nevertheless, it is within the present invention thatfurther miRNAs are deleted from the genome of the beta-herpesvirus ofthe present invention which is advantageous for the beta-herpesvirus ofthe present invention. Thus in an embodiment of the beta-herpesvirusaccording to the present invention wherein the beta-herpesviruscomprises the deletion of at least one miRNA, the miRNA is preferablyselected from the group comprising miRNAs having immune modulatordfunctions (Dölken L et al., J Virol. 2007).

In order to provide guidance to a person skilled in the art on how abeta-herpesvirus of the present invention is designed, and in particulara beta-herpesvirus of the present invention wherein an immunemodulatorygene is deleted from the genome of the beta-herpesvirus, and how suchimmune modulatory gene may be selected, the present inventor conceived avaccine according to the present invention by inserting the NKG2D ligandRAE-1γ into the MCMV genome in place of the m152 gene, that otherwisenegatively regulates this NKG2D ligand. Accordingly, said virusrepresents an embodiment, wherein an immune modulatory gene has beendeleted the gene product of which results in preventing theaccessibility of an NKG2D ligand to the cell surface.

Whitout wishing to be bound by any theory the present inventor currentlyassumes that: the deletion of an immunomodulatory gene, whichdownregulates the cellular ligand, (a) prevents downregulation of bothendogenous cellular ligand and cellular ligand encoded by thebeta-herpesvirus of the present invention, in this particular exampleRAE-1γ and RAE-1γ encoded by the MCMV; (b) may override the consistentexpression of the cellular ligand, in this particular example RAE-1γ, oninfected cells the effect of all other CMV immunoevasins encoded byimmune modulatory genes, for NK cells, and (c) may also augment CD8⁺ Tcell response through co-stimulatory function of NKG2D on these cells.

Furthermore, since m152 additionally arrests the maturation of MHC classI molecules (Ziegler, H. et al., 1997, Immunity 6:57-66), the deletionof this gene may improve the presentation of viral proteins and enhancethe T cell immune response. Herein the present inventor demonstratesthat RAE-1γ-expressing MCMV, also referred to herein as RAE-1γMCMV, isdramatically attenuated in vivo not only in the immunocompetent host buteven in immunologically immature neonata mice and in immunodeficientmice. However, despite tight immune control, RAE-1γMCMV infectionelicited a potent, long-lasting cellular and antibody immune responseable to protect animals against challenge infection. Moreover, maternalRAE-1γMCMV infection resulted in the production and placental transferof antiviral antibodies that protected their offspring from MCMVinfection following neonatal infection.

In an embodiment of the beta-herpesvirus according to the presentinvention, wherein the beta-herpesvirus is suitable for inducing animmune response, the immune response is preferably an immune responseagainst a beta-herpesvirus. In a further preferred embodiment the immuneresponse preferably comprises neutralizing antibodies againstbeta-herpesvirus and/or CD4⁺ T-cells directed against epitopes ofbeta-herpesvirus and/or CD8⁺ T-cells directed against epitopes ofbeta-herpesvirus. In connection therewith it is important to understandthat the present invention considers that in connection with an immuneresponse against a beta-herpesvirus, preferably comprising neutralizingantibodies directed against a beta-herpesvirus and/or CD4⁺ T-cellsdirected against epitopes of beta-herpesvirus and/or CD8⁺ T-cellsdirected against epitopes of beta-herpesvirus, the beta-herpesvirus isnot necessarily from the same beta-herpesvirus strain and/orbeta-herpesvirus species as the beta-herpesvirus suitable anc actuallyfor inducing and elicting, respectively, an immune response. A personskilled in the art will acknowledge that different strains of onespecies of beta-herpesvirus such as different HCMV strains is preferablyused as the beta-herpesvirus according to the present invention iscapable of and/or suitable for inducing an immune response, wherein theimmune response is directed against a beta-herpesvirus of anotherstrain, such as another HCMV strain. A person skilled in the art willalso acknowledge that the infection with beta-herpesviruses is generallythought to be species-specific. In other words, HCMV is only capable ofinfecting humans, MCMV is only capable of infection mice. Nevertheless,it is also possible that a beta-herpesvirus species may infect morerelated species, such as MCMV is capable of infecting other rodents thanmouse, or rhesus CMV is capable of infecting humans. It is also withinthe present invention that such species-barrier will be overcome, e.g.by genetic manipulation with regard to entry of the virus, receptors,glycoproteins expressed at the surface and/or prevention of apoptosis.Having said this it is an embodiment of the present invention thatimmune response against a beta-herpesvirus, preferably the neutralizingantibodies and/or CD4⁺ T-cells and/or CD8⁺ T-cells, is directed againstthe beta-herpesvirus capable of and/or suitable for inducing an immuneresponse, wherein the beta-herpesvirus suitable for inducing an immuneresponse is of the same strain and species, of the same species butdifferent strain and/or from a different species and different strain asthe beta-herpesvirus the immune response is directed against. In a morepreferred embodiment the beta-herpesvirus suitable for inducing animmune response induces an immune response preferably comprisingneutralizing antibodies against the beta-herpesvirus and/or CD4⁺ T-cellsdirected against epitopes of the beta-herpesvirus and/or CD8⁺ T-cellsdirected against epitopes of the beta-herpesvirus, wherein thebeta-herpesvirus preferably is suitable for inducing an immune responseis HCMV.

The present specificaton also provides evidence that expression of acellular ligand such as an NKG2D ligand on beta-herpesvirus infectedcells dramatically attenuates the virus growth in vivo but in the sametime does not lead to attenuation in vitro. It is immediately understoodthat a person skilled in the art will prefer a virus which is attenuatedin vivo as attenuation is generally understood to be conterminous withsafety (Cicin-Sain, L. et al., supra). Accordingly, in an embodiment ofthe beta-herpesvirus according to the present invention thebeta-herpesvirus of the present invention is attenuated in vivo.

Attenuation as used herein preferably means that a virus such as a liveattenuated virus, such as the beta-herpesvirus of the present invention,is replicating to lower titers compared to the respective wild typevirus. Attenuation as used herein more preferably means that a virus hasa virulence which is reduced compared to the respective wild type virus.Attenuation alters a virus so that it becomes more harmless and/or lessvirulent. These vaccines contrast to those produced by “killing” thevirus (inactivated vaccine). Still more preferably, attenuation as usedherein means that the titer of a virus is less compared to the titer ofthe respective wild type virus grown under the same conditions. So as todetermine whether the beta-herpesvirus according to the invention andparticularly the HCMV according to the present invention is attenuatedcompared to the respective wild type virus, preferably, the assay forassessing virus growth kinetics as described in Example 1 is used. It isknown to a person skilled in the art that in connection with a titer ofa virus the term “plaque-forming units” also referred to herein as PFU,is used.

The titer of a virus in terms of “plaque-forming units” is preferably ameasure for the amount of infectious virus particles present in asample, such as a sample from a subject or an in vitro sample. Todetermine virus titer a standard plaque assay as for example describedin Krmpotic, A. et al. (Krmpotic, A. et al. 2005, supra) and/ordescribed in Example 1 is used. A person skilled in the art willfurthermore know how to determine the amount of a pathogen, such as thetiter of a virus, in a subject or in an in vitro sample. The amount ofbacteria, such as Listeria monocytogenes, in a sample or subject ispreferably measured as “colony forming units”, also referred to hereinas CFU. To determine CFU a person skilled in the art will knowappropriate assays. As an example an assay to determine CFU in mouseorgans is preferably an assay as described in Example 1 herein.

In connection with attenuation it will be furthermore understood that aperson skilled in the art will regard the attenuation of a virus as ameasure for the safety of the virus if used as a vaccine or vaccinevector. Accordingly, it is advantageous with regard to safety if a virusis attenuated. Such attenuation is preferably achieved in many waysknown to a person skilled in the art. For example the deletion of one ormore immune modulatory genes preferably results in attenuation of avirus in vivo, as the immune response is stronger if the immunemodulatory gene encoding a gene product down-modulating and/ordown-regulating the immune response is deleted (Cicin-sain, L. et al.,supra). It will be also understood that the strongest possible deletionis the absence of virus replication and/or virus progeny. Such moststrongest attenuation will be achieved if an essential gene is deletedfrom the virus (Mohr, C. A. et al., 2010, supra).

Accordingly, it will be understood that the strongest conceivable degreeof attenuation is the formal impossibility of a virus to finish itsreplication cycle and/or incits inability of generating viral progenyand/or incapable of generating infectious viral progeny. Such virusincapable of finishing its replication cycle and/or generating viralprogeny and/or incapable of generating infectious viral progeny, isknown in the art as spread-deficient and/or replication deficient virusas described for example in (Mohr, C. A. et al., 2010, supra; Snyder, C.M. et al., 2009, J Immunol, 182:128.29). In an embodiment of thebeta-herpesvirus of the present invention the beta-herpesvirus isdeficient in at least one gene product encoded by an essential geneand/or by a glycoprotein, wherein the deficiency of said essential geneand/or glycoprotein results in a virus which is incapable of replicatingits viral genome, spreading its progeny from one cell to another and/orproducing infectious viral progeny. Such deficiency is achieved in anembodiment of the present invention by deletion or partial deletion ofthe coding sequence of the respective gene, or by inhibition of thetranscription, translation or function thereof. Respective genes areknown to the person skilled in the art. By way of example, a virushaving a deletion of a gene encoding a glycoprotein essential forinfection is disclosed in Snyder et al., (Snyder, C. M. et al., 2009,supra) and a virus having a deletion of a gene encoding a protein beingessential for virus spread is disclosed in Mohr et al. (Mohr, C. A. etal., 2010, supra). Such virus is preferably produced using a cell linewhich is capable of complementing the gene product of the deletedessential gene and/or glycoprotein. The generation and use of such celllines is known to the person skilled in the art.

However, in spite of reduced antigenic load due to attenuation of thebeta-herpesvirus of the present invention such as CMV expressing Rae-1γ,a beta-herpesvirus of the present invention is capable of inducing astrong immunity and protect immunocompetent subjects as well asimmunodeficient subjects against infection with wild typebeta-herpesvirus.

It is important to note that the present inventor provides conclusiveevidence in the Examples shown herein that subjects immunized with thebeta-herpesvirus of the present invention are able to resist challengeinfection better than subjects immunized with the respective wild typebeta-herpesvirus. In connection therewith it is important to understandthat it is known for decades that infection with wild typebeta-herpesvirus, especially with MCMV wild type, which is not useful asa vaccine for reasons of safety, confers protection against infectionwith wild type virus, preferably of the same strain.

Most importantly, the present inventor found that in naturally moreresistant C57BL/6 mice immunized with the beta-herpesvirus of thepresent invention, more particularly RAE-1γMCMV, the CD8⁺ T cellresponse is stronger compared to individuals infected with therespective wild type virus. Newborn subjects derived from femalesimmunized with the beta-herpesvirus of the present invention areprotected against perinatal infection to the same extent as theoffspring derived from females immunized with wild type virus. Thislatter finding confirmed that the beta-herpesvirus of the presentinvention induces neutralizing antibody response despite strongattenuation and therefore lower antigenic load. More particularly,protection of newborn individuals against challenge infection issmediated through transplacentally transferred IgG.

Another important characteristic of the beta-herpesvirus of the presentinvention is its ability to provide long-lasting immunity. Although theload of latent viral genome is significantly lower in subjects immunizedwith the beta-herpesvirus of the present invention this virus is able toreactivate upon immunosuppression of latently infected subjects.Notably, even though the beta-herpesvirus according to the presentinvention is under strong selective pressure by NK cells and CD8⁺ Tcells, the present inventor has not observed any mutation of theinserted gene encoding the cellular ligand. More importantly, all theviruses recovered from latency showed no differences in comparison withparental virus mutant used for primary infection.

It is the merit of the present inventor having recognized that thebeta-herpesvirus of the present invention which comprises at least oneheterologous nucleic acid, wherein the at least one heterologous nucleicacid comprises a gene encoding a cellular ligand, is a suitable vaccinevector. This prompted the present inventor to insert immunodominantantigens of different pathogens into the beta-herpes virus in order totest its vector capacity in terms of eliciting an immune response in therecipient organism against said immunodomiunant antigens.

The present inventor unexpectedly found that when the beta-herpesvirusaccording to the present invention encodes an additional heterologousnucleic acid, wherein the additional heterologous nucleic acid codes fora peptide, oligopeptide, polypeptide and/or protein which constitutes orcomprises at least one antigen and epitope, respectively, that saidbeta-herpesvirus was dramatically attenuated in vivo:

In connection therewith it is known to the person skilled in the artthat NK cells have a major role in innate immune response to severalviruses. Receptors, such as NKG2D, are among the most potent activatingreceptors expressed on all NK cells and all CD8⁺ T cells in humans, oractivated CD8⁺ T cells in mice. Insertion of a gene encoding a cellularligand, for example NKG2D ligand RAE-1γ, in place of its viraldownregulator, i.e. an immune modulatory gene such as, for example m152,results in dramatic virus attenuation. Attenuation as such is notunexpected, since even the mutant viruses lacking viral inhibitors ofthe cellular ligand(s) are attenuated (Krmpotic, A. et al., 2002, supra;Krmpotic, A. et al., 1999, J Exp Med,190(9):1285-96; Arapovic, J. etal., 2009, supra; Hasan, M. et al., 2005, J Virol, 79:2920-30; Lenac, T.et al., 2006, supra; Lodoen, M. et al., 2003, J Exp Med,197(10):1245-53; Lodoen, M. B. et al., 2004, J Exp Med 200(8):1075-81),but in case of the beta-herpesvirus of the present invention expressingthe cellular ligand, said beta-herpesvirus of the present invention, inan embodiment, is attenuated to such an extent that it even fails toreach salivary gland. A person skilled in the art will immediatelyacknowledge that salivary glands are otherwise a privileged site forvirus persistency and horizontal spread. The results obtained in miceexpressing Ly49H receptor, otherwise resistant to MCMV because ofrecognition of viral m157 protein, also demonstrate dramaticattenuation, confirming that this can not be only the consequence of anNK cell response. More particularly, in these mice NK cells are stronglyactivated through Ly49H/m157 interaction. Moreover, the beta-herpesvirusof the present invention expressing a cellular ligand is attenuated inotherwise severely immunocompromized subjects, for example mice lackingIFNAR, mice immunodepleted by sublethal gamma irradiation, and micelacking perforin. These latter findings are extremely important bearingin mind that the hallmark of beta-herpesviruses is an opportunisticinfection in immunocompromised individuals.

The present inventor furthermore found that when the beta-herpesvirusaccording to the present invention encodes an additional heterologousnucleic acid, wherein the at additional heterologous nucleic acidpreferably codes for a peptide, oligopeptide, polypeptide and/or proteinwhich more preferably constitutes or comprises at least one antigen andepitope, respectively, an unexpected strong CD8⁺ T cell response inspite of virus attenuation is detected, whereby preferably such stronCD8⁺ T cell response is directed to said antigen and epitope,respectively.

In connection therewith a person skilled in the art will acknowledgethat it is a current dogma in the field of vaccination that a strong NKcell response after primary virus infection should result incompromised, i.e. weaker, CD8⁺ T cell response. This theory is supportedby recent work by Andrews et al. (2010) who reported that NK cellresponse has negative effect on generation of virus specific CD8⁺ T cellresponse by restricting duration of exposure of T cells to infectedantigen-presenting cells. These findings are supported by recent work inC57BL/6 mice infected with WT-MCMV, which expresses m157, or viruslacking m157 (Mitrovic, M. et al, under revision). Working on a tumormodel, Soderquest, K. et al.) reported that NK cells kill recentlyactivated CD8⁺ T cells and thus lead to a less efficient CD8⁺ T cellresponse (Soderquest, K. et al., 2011, Blood., 117(17):4511-8.).Therefore, based on the above findings, one would expect that abeta-herpesvirus of the present invention would induce less potent CD8⁺T cell response, particularly because of lower antigenic load. Incontrast to the expectations of a person skilled in the art the presentinventor has shown that mice infected with RAE-1γMCMV demonstrated equalor even more potent CD8⁺ T cell response against immunodominant MCMVepitopes. Subjects infected with the beta-herpesvirus of the presentinvention demonstrated equal or even more potent CD8⁺ T cell responseagainst immunodominant beta-herpesvirus epitopes. Moreover, the abovementioned results were confirmed and extended by using thebeta-herpesvirus of the present invention wherein the beta-herpesviruscomprises at least one heterologous nucleic acid, wherein the at leastone heterologous nucleic acid comprises a gene encoding a cellularligand, as a vector of foreign antigens such as, for example, HA ofinfluenza or listeriolysin epitope of Listeria monocytogenes. Whenconducting experiments the present inventor found that CD8⁺ T cellresponse is much stronger in case of a beta-herpesvirus of the presentinvention expressing a cellular ligand in addition to foreign antigenssuch as viral, bacterial and/or parasite antigens, as compared to abeta-herpesvirus not expressing said cellular ligand.

In an embodiment of the beta-herpesvirus according to the presentinvention, wherein the beta-herpesvirus expressing a cellular ligand inaddition to foreign antigens such as viral, bacterial and/or parasiteantigen the antigen is an antigen specific for a virus, bacterium and/orparasite, respectively.

In an embodiment of the beta-herpesvirus according to the presentinvention the antigen is an antigen specific for Listeria, preferablyselected from the group comprising listeriolysin O (LLO), whereby thebeta-herpesvirus of the present invention then is useful for thetreatment and/or prevention of listeriosis. In connection therewith thepresent inventor has surprisingly found that the beta-herpesvirusaccording to the present invention confers an unexpected strongprotection against challenge infection with Listeria monocytogenes

Subjects infected with a wild type beta-herpesvirus expressinglisteriolysin epitope, i.e. a beta-herpesvirus wherein thebeta-herpesvirus does not comprise a heterologous nucleic acid, whichcomprises a gene encoding a cellular ligand; and subjects infected withthe beta-herpesvirus of the present invention expressing listeriolysinboth developed antilisteriolysin CD8⁺ T cell response, although thefrequency of listeriolysin specific CD8⁺ T cells was higher afterinvention with the beta-herpesvirus of the present invention. However,when these subjects were challenged with sublethal and lethal doses ofListeria monocytogenes the protection was far better in the group ofsubjects vaccinated with the beta-herpesvirus of the present inventionexpressing listeriolysin. The enhanced protection was demonstrated notonly with respect to reduction of bacterial load but also with regard toreduced tissue pathology. Since both groups of subjects were vaccinatedand had listeriolysin specific CD8⁺ T cells, such dramatic differenceswith respect to protective capacity was absolutely unexpected.

MCMV infection caused depletion of conventional dendritic cells (Andrewset al., 2001, Nat Immunol, 2(11):1077-84.). However, the presentinventor has found that in subjects infected with the beta-herpesvirusof the present invention expressing a cellular ligand, the frequency ofconventional DCs was preserved in comparison to wild typebeta-herpesvirus infection. In connection therewith a person skilled inthe art will acknowledge that the results obtained when using thebeta-herpesvirus of the present invention are extremely surpsising. Thebeta-herpesvirus of the present invention allows a potent CD8⁺ T cellresponse in spite of virus attenuation.

Finally, the present inventor has surprisingly found that the geneencoding a cellular ligand expressed in context of beta-herpesvirusinfection was not subject of deletions or mutations, genetic changes,due to the selective pressure by strong immune response.

It is well established that in Ly49H⁺ mice the MCMV gene encoding m157is subject to intense genetic changes, such as mutations and deletions,resulting in viruses which are no longer recognized by Ly49H and nolonger sensitive to NK cells. After observing dramatic attenuation ofthe beta-herpesvirus of the present invention expressing a cellularligand, the present inventor is currently of the opinion without wishingto be bound thereby that such intense genetic changes according to whathas been said above will also appear in connection with thebeta-herpesvirus of the present invention, i.e. that under selectivepressure by NK cells via receptors, the gene encoding the cellularligand will be the subject to mutations. Therefore the present inventorperformed reactivation of latent beta-herpesvirus of the presentinvention by depleting subjects of T cells and NK cells. Surprisingly,although the beta-herpesvirus of the present invention was able toreactivate from latency, in 72 isolated clones of recurrentbeta-herpesvirus of the present invention no mutation could be observed.Therefore, all viruses according to the present invention preserved anintact gene enconding cellular ligand. The present inventor deems thisto be very important, because this seems to contribute to a more potentprotective capacity long after initial priming, vaccination.

So as to determine whether the beta-herpesvirus of the present inventionand particularly the HCMV of the present invention is expressing acellular ligand, preferably, the assay as described in Example 2 isused. More precisely, the expression of a cell receptor ligand can beassessed by Western blot with an antibody specific for the cellularligand and/or by FACS analysis using preferably a monoclonal antibodyspecific for the cellular ligand. In order to demonstrate that theimmune response is enhanced in subjects which are infected with abeta-herpesvirus of the present invention an NK cell assay, preferablyaccording to the NK cell assay described in the examples herein can beused to demonstrate susceptibility of the recombinant beta-herpesvirusof the present invention to NK cells. By blocking the receptor on immunecells, such as DC, or the cellular ligand on cells infected with thebeta-herpesvirus according to the present invention, the essential roleof signaling in enhanced NK cell response is demonstrated. T cellresponse to either beta-herpesvirus antigen(s) or antigens comprised inor encoded by an additional heterologous nucleic acid can be assessed byvarious means, preferably by determining the frequency of T cellsspecific for the antigen(s) and/or the epitope(s) by FACS analysisand/or by ELISPOT assay.

More specifically, the expression of the NK cell receptor ligand ULBP2is preferably assessed by Western blot analysis using human ULBP-2antibody (R&D Systems; cat. No. AF1298) and/or by FACS analysis withmonoclonal anti-human ULBP-2-PE antibody (R&D Systems; cat. No.FAB1298P).

The assay used herein in conecction with various mouse experiments inorder to demonstrate enhanced immune response in mice infected withRae-1MCMV or additional viruses comprising, in addition to Rae-1, anadditional heterologous nucleic acid(s), more preferably expressingantigen(s) can be applied for respective HCMV viruses, such as HCMVexpressing ULBP2:

1. NK cell assay can be used to demonstrate NK cell susceptibility ofthe recombinant virus. By blocking NKG2D receptor on NK cells or ULBP2on infected cells one can demonstrate the essential role of NKG2Dsignaling in enhanced NK cell response.

2. T cell response to either HCMV antigens or foreign antigens in caseof HCMV expressing cellular ligand, such as ULBP2, virus as a vector,can be assessed by various means, including determination of frequencyof T cells specific for foreign antigen (epitope) by FACS or by ELISPOTassay.

In an embodiment of the beta-herpesvirus of the present invention thebeta-herpesvirus is used as a vaccine and/or vector. In a furtherembodiment thereof the beta-herpesvirus encodes for a heterologousnucleic acid. Preferably such heterologous nucleic acid codes for anantigen and epitope, respectively, more preferably an antigen andepitope, respectively, of a pathogen. Because of this such vaccine andvector, respectively, is suitable for the treatment and/or prevention ofa disease caused by or associated with said pathogen. Such pathogenspreferably comprise viruses, parasites and bacteria. In an embodimentthe antigen is an antigen specific for influenza and is preferablyselected from the group comprising full-length form of HA of Influenzaand headless form of HA of Influenza, neuraminidase and nucleo-protein,whereby the beta-herpesvirus of the present invention expressing suchantigen is then useful for the treatment and/or prevention of influenza.In connection therewith it will be understood that the headless form ofHA of Influenza as used herein preferably means the form of HA ofinfluenza without the globular head domain in the HA1 subunit of thehemaglutinin. Hemaglutinin (HA) is the major glycoprotein from InfluenzaA virus (strain A/Puerto Rico/8/1934 H1N1) Furthermore, HA is anintegral membrane glycoprotein comprised out of two subunits: HA1 andHA2. Given the fact that HA2 subunit of the influenza virushemagglutinin is relatively well conserved, but during natural infectionor vaccination with conventional influenza vaccine masked by highlyimmunogenic globular head domain of HA. It is assumed that theHCMV-ULBP2 HA headless vaccine will elicit cross-reactive anti-HA2antibodies. It is further assumed that a group of subjects vaccinatedwith HCMV-ULBP2 HA headless vaccine will develop serum neutralizinganti-HA2 antibodies prior than the placebo group and will developanti-HA2 CD8⁺ T cells in the peripheral blood samples prior to thecontrol group. Also, it is assumed that vaccination with HCMV-ULBP2 HAfull-length and HCMV-ULBP2 HA headless vaccine will providecross-protectively against seasonal influenza outbreaks. In connectiontherewith it is important to note that the vaccination of mice withheadless HA elicits immune sera with broader reactivity than thoseobtained from mice immunized with a full-length HA (Steel, J. et al.,2010, mBio, 1(1):e00018-10). The Headless HA construct as used herein ispreferably a HA immunogen comprising the conserved influenza HA stalkdomain (HA2 subunit) and lacking the globular head. Without wishing tobe bound by any theory the present inventors assume further that theconserved stalk HA2 subunit is masked by the highly immunogenic globularhead domain in the HA1 subunit during natural infections. Constructionof headless HA is described by Steel et al., 2010, supra. In brief,headless HA lacks the globular head domain flanked by conserveddisulfide bond linking cysteines 52 and 277 of HA1, which is replacedwith a GGGG linker.

In a further embodiment the antigen is an antigen specific forMycobacterium tuberculosis which is preferably selected from the groupcomprising Antigen 85A (Accession No. P0A4V2; A85A_MYCTU; Sander, C. R.et al., 2009, Am J Respir Crit Care Med. 2009; 179(8): 724-733), Antigen85B (Accession No. P0C5B9), -ESAT-6 (Accession No. P0A564 (ESXA_MYCTU))(A85B_MYCTU)(Brandt, L. et al., 2000, Infect Immun, 68(2):791-5.) andAntigen 85B-TB10.4 (Accession No. P0A568 (ESXH_MYCTU))(Dietrich, J. etal., 2005, J Immunol, 174(10):6332-9), whereby the beta-herpesvirus ofthe present invention expressing such antigen is then useful for thetreatment and/or prevention of tuberculosis.

In a still further embodiment the antigen is an antigen specific forPlasmodium and is preferably selected from the group comprisingcircumsporozoite (CS) protein, wherein Plasmodium is preferably selectedfrom the group comprising Plasmodium falciparum, Plasmodium vivax,Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi, wherebythe beta-herpesvirus of the present invention expressing such antigen isthen useful for the treatment and/or prevention of malaria. In a stillfurther embodiment the antigen is an antigen specific for HIV and ispreferably selected from the group comprising HIV-1 gag(NC_(—)001802.1), whereby the beta-herpesvirus of the present inventionthen is useful for the treatment and/or prevention of HIV. In a stillfurther embodiment the antigen is an antigen specific for Listeria,whereby the antigen is preferably selected from the group comprisinglisteriolysin O (LLO), whereby the beta-herpesvirus of the presentinvention expressing such antigen is then useful for the treatmentand/or prevention of listeriosis.

In a still further embodiment the antigen is an antigen specific forRespiratory syncytial virus and is preferably selected from the groupcomprising glycoprotein F (JN032120.1) and glycoprotein G (JN032120.1),whereby the beta-herpesvirus of the present invention expressing suchantigen is then useful for the treatment and/or prevention ofRespiratory syncytial virus. Respiratory syncytial virus is alsoreferred to herein as RSV. In connection therewith it will be understoodthat various forms of F and G glycoproteins as well as not known F or Gepitopes can be used as antigens to be expressed by the beta-herpesvirusof the present invention. It will be also understood that epitoperecognition differs among individual human HLA types. In connectiontherewith the present inventors assume without wishing to be bound byany theory that by introduction of different forms of F and Gglycoproteins and their expression in the context of thebeta-herpesevirus of the present invention, infection whith suchbeta-herpesvirus will provide wider protectivity and thus elicitstronger immune responses against RSV strains.

In an embodiment of the beta-herpesvirus of the present inventionwherein the antigen is an antigen specific for Respiratory syncytialvirus, the antigen is preferably selected from the group comprisingfull-length glycoprotein F, soluble glycoprotein F, full-lengthglycoprotein G, codon-optimized full-length glycoprotein F,codon-optimized soluble glycoprotein F and codon-optimized full-lengthglycoprotein G, wherein the glycoproteins are preferably of the RSV A2strain. In an embodiment of the beta-herpesvirus of the presentinvention wherein the antigen is an antigen specific for Respiratorysyncytial virus, the antigen is preferably selected from the groupcomprising glycoprotein F (D53); full-length glycoprotein F (D53);soluble glycoprotein F (D53); soluble glycoprotein F (D53), wherein aself-trimerizing peptide is fused to the C-terminus; solubleglycoprotein F (D53), wherein both cleavage sites are mutated so that itis not cleaved; soluble glycoprotein F (D35), wherein both cleavagesites are mutated so that it is not cleaved and wherein aself-trimerizing peptide is fused to the C-terminus; solubleglycoprotein F (D35), wherein 10 amino acids of the fusion peptide aredeleted.

In a still further embodiment of the beta-herpesvirus according to thepresent invention the antigen is an antigen specific for HPV and ispreferably selected from the group comprising E6 29-38 (TIHDIILECV;restricted by the HLA-A0201 molecule), E6 29-37 (TIHDIILEC; restrictedby B48), and E6 31-38 (HDIILECV; restricted by B4002), E6 52-61(FAFRDLCIVY; restricted by B57), E6 and E7 (JN171845.1) (Nakagawa, M. etal., 2007, J Virol, 81(3):1412-23), whereby the beta-herpesvirus of thepresent invention expressing such antigen is then useful for thetreatment and/or prevention of HPV.

In a still further embodiment the antigen is an antigen specific forhelicobacter pylori and is preferably selected from the groupcomprisingurease, VacA, CagA, heat shock protein, neutrophil-activatingprotein outer membrane lipoprotein, babA2 (Kimmel, B. et al., 2000,Infect Immun, 68(2):915-20; Yamaoka, Y. et al., 2008, World JGastroenterol, 14(27): 4265-4272), whereby the beta-herpesvirus of thepresent invention expressing such antigen is then useful for thetreatment and/or prevention of helicobacter pylori.

In the embodiments of the present invention wherein the beta-herpesvirusof the present invention encodes at least one additional heterologousnucleic acid, wherein the at least one additional heterologous nucleicacid is a heterologous nucleic acid coding for a peptide, oligopeptide,polypeptide or protein, wherein the peptide, oligopeptide, polypeptideor protein constitutes or comprises at least one antigen and epitope,respectively, the antigen and epitope, respectively, is preferably animmunodominant antigen. In a further embodiment the expression of saidantigen and epitope, respectively, is sufficient for inducing an immuneresponse, preferably an immune response protective against naturalinfection with the pathogen comprising said antigen and epitope,respectively.

In a further embodiment of the beta-herpesvirus according to the presentinvention, an/the additional heterologous nucleic acid comprised in thebeta-herpesvirus of the present invention is less than ˜4 kb.

In a preferred embodiment of the beta-herpesvirus according to thepresent invention, the additional heterologous nucleic acid comprises apromoter, preferably a viral promoter, capable of expressing the antigenwith kinetics selected from the group comprising immediate-earlykinetics and early kinetics, preferably immediate-early kinetics. Suchpromoter is preferably selected from the group comprising an IEpromoter. The present inventor assumes without wishing to be bound byany theory that expression of the antigen with immediate-early kineticswill lead to a boost of the immune response independent of thegeneration of infectious progeny.

A person skilled in the art will acknowledge that it is preferred thatthe antigen is properly processed in order to guarantee transport andpresentation by MHC class I molecules.

In a further embodiment of the beta-herpesvirus according to the presentinvention the beta-herpesvirus encodes one or more than one additionalheterologous nucleic acid(s). In connection therewith it is whitin anembodiment of the beta-herpesvirus according to the present inventionthat the one or more than one additional heterologous nucleic acid(s)code(s) for a peptide, oligopeptide, polypeptide or protein. In afurther embodiment the one or more than one heterologous nucleic acid(s)codes for one or more than one peptide(s), one or more than oneoligopeptide(s), one or more than one polypeptide(s) and/or one or morethan one protein(s). It is a further embodiment that the peptide(s),oligopeptide(s), polypeptide(s) and/or protein(s) constitute(s) orcomprise(s) one or more than one antigen(s) and epitope(s),respectively. Accordingly, the beta-herpesvirus of the present inventionis preferably suitable to or capable of inducing an immune responseagainst beta-herpesvirus, and additionally is suitable to or capable ofinducing an immune response against the one or more than one peptide(s),the one or more than one oligopeptide(s), the one or more than onepolypeptide(s) and/or the one or more than one protein(s). In anembodiment the beta-herpesvirus of the present invention is suitable toor capable of inducing an immune response against one or morepathogen(s) having the one or more than one antigen(s).

In the embodiments of the beta-herpresviruses of the present inventionwhere the beta-herpesvirus expresses one or more than one antigenderived from one or more than one pathogen(s) the beta-herpesvirus issuitable to or capable of inducing an immune response against therespective pathogen an antigen of which is expressed by thebeta-herpesvirus according to the present invention. For example abeta-herpesvirus of the present invention is suitable to induce animmune response against beta-herpesvirus, is suitable to induce animmune response against beta-herpesvirus and influenza, is suitable toinduce an immune response against beta-herpesvirus and listeria, or issuitable to induce an immune response against beta-herpesvirus,influenza and listeria. It is important to understand that each and anyantigen suitable to induce an immune response against the respectivepathogen is preferably combined with each and any other antigen suitableto induce an immune response against the same or different pathogen inorder to generate a beta-herpesvirus of the present invention preferablybeing a vaccine against each and any of the above mentioned pathogens.

In a further embodiment wherein more than one antigen is encoded theseantigens are preferably derived from one pathogen. Accordingly abeta-herpesvirus of the present invention is suitable to induce animmune response against beta-herpesvirus and against more than oneantigen and epitope, respectively, of the respective pathogen. Inconnection therewith it is an embodiment to introduce different epitopesof one pathogen, preferably immunodominant epitopes of one pathogen intothe genome of the beta-herpesvirus. In a further embodiment more thanone epitope of different species and/or strains of the pathogen areintroduced into the beta-herpesvirus genome. For example, differentstrains of mycobacterium will exhibit different epitopes, accordinglysaid different epitopes derived from different strains will be comprisedin the genome of the beta-herpesvirus of the present invention.

A person skilled in the art will acknowledge that in connection withvarious embodiments antigens displaying a limited variability arepreferred. More preferably, antigens having conserved domains are used.It will also be understood immediately that if a peptide is consideredas an antigen, the selection of said peptide will depend on HLArestriction.

In connection with the various embodiments of the beta-herpesvirus ofthe present invention, the beta-herpesvirus is preferably a CMV, morepreferably an HCMV.

It will be immediately understood by a person skilled in the art thatmore than one strain of a particular beta-herpesvirus exists. Saidstrains exhibit different characteristics in terms of degree ofattenuation, tropism and/or genes comprised in the genome. In connectiontherewith it is preferred that the beta-herpesvirus of the presentinvention is attenuated due to genetic manipulation, and ischaracterized by showing expression of a cellular ligand, deletion of animmunemodulatory gene, deletion of miRNA, deletion of a non-essentialgene other than immunemodulatory gene and/or deletion of an essentialgene. In a further embodiment the beta-herpesvirus of the presentinvention has a tropism like a wild type beta-herpesvirus strain.

A wild type beta-herpesvirus strain, as preferably used herein meansthat the virus is a beta-herpesvirus strain which has been isolated fromits native host and which preferably has maintained its ability toinfect endothelial cells, epithelial cells, macrophages and DC in tissueculture. Accordingly, a wild type CMV strain as preferably used hereinmeans that the virus is a CMV strain which has been isolated from itsnative host and which preferably has maintained its ability to infectendothelial cells, epithelial cells, macrophages and DC in tissueculture. With regard to CMV tropism it is also referred to thepublications of Sinzger C et al. (Sinzger, C. et al., 1996,Intervirology; 39(5-6):302-19; Sinzger, C. et al., 2008, Curr TopMicrobiol Immunol; 325:63-83).

Examples for HCMV clinical isolates are, among others TB40/E, which wasisolated from a patient by throat wash of a bone marrow transplantrecipient and which was propagated for 5 passages in fibroblast,followed by 22 passages in endothelial cells (Sinzger, C. et al., 1999,J Gen Virol, 80(Pt 11):2867-77); the Towne strain which derived fromurine of a congenitally infected newborn, which was attenuated through125 passages in human embryonic lung fibroblast cell cultures; andAD169, which was originally recovered from adenoid tissue (Rowe, W. P.et al., 1956, Proc Soc Exp Biol Med, 92:418-424) and which has beenextensively passaged in human fibroblast cell cultures and lost itsnatural cell tropism for endothelial cell.

In the case of laboratory-adapted HCMV-strain AD169 or attenuated Townestrain which were extensively propagated in human embryonic lungfibroblasts (HELF) both strains lost their endothelial cell tropism andleukotropism. However, inoculation of such strains in human umbilicalvein endothelial cells (HUVEC) restored its endothelial tropism asconfirmed by immunofluorescence using monoclonal antibodies against HCMVproteins (pp 65, IE-1 and gB), or by determination of viral titer andleukotropism as confirmed by standard assays for PMNL andmonocyte-tropism as reported (Gerna, G. et al., 2002, J Gen Virol, 83(Pt8):1993-2000.). PMNL were isolated from healthy donors and coculturedwith infected HUVEC, were subsequently placed in the upper compartmentof a cell culture device separated with a transwell filter from thelower compartment with FMLP which attracts PMNLs. Infection wasdetrmined by immunofluorescence using monoclonal antibodies against HCMVproteins (pp 65, IE-1 and gB), or by determination of viral titer.

In a preferred embodiment the beta-herpesvirus of the present inventionis capable of infecting professional antigen presenting cells,preferably comprising dendritic cells and macrophages. In a furtherpreferred embodiment the beta-herpesvirus of the present invention iscapable of infecting endothelial cells, epithelial cells andfibroblasts. It is a further embodiment of the beta-herpesvirus of thepresent invention that the capability to infect certain cell types islimited. Such limitation of the capability to infect certain cell typesresults for example from genetic manipulation. For example if abeta-herpesvirus is deficient in at least one receptor for the infectionof a certain cell type, such as a certain glycoprotein, such virus willnot be able to infect the respective cell type. In a further embodimenta gene coding for a receptor for the infection of a certain cell type isintroduced into the genome of the beta-herpesvirus of the presentinvention. In such case, expression of such receptor from the genome ofthe beta-herpesvirus, e.g. the receptor mediating infection ofendothelial cells and/or dendritic cells, will result in abeta-herpesvirus preferentially infecting the respective cell types. Ina further preferred embodiment the capability to infect certain celltypes of the beta-herpesvirus is limited to certain cell types, wherebyin addition a gene coding for a receptor for the infection of thecertain cell type is introduced into the genome of the beta-herpesvirusof the present invention. Such virus will preferentially infect thecell-types via the receptor for the infection of the certain cell type.In connection therewith it is important to note that beta-herpesvirusesare able to infect professional antigen presenting cells. In anembodiment the beta-herpesvirus of the present invention is capable ofinfecting professional antigen presenting cells. In a further embodimentthe beta-herpesvirus of the present invention is capable of infectingprofessional antigen presenting cells only or with high preference. Inconnection therewith it is important to note that a hallmark of HCMVinfection is the targeting of monocytes. It will be acknowledged by aperson skilled in the art that the HCMV genome is maintained even in theabsence of a productive infection. In connection therewith, monocytesare considered to be reservoirs for viral genome involved in the viralspread and most importantly that their immunomodulation facilitates HCMVsurvival and avoidance of an immune response. HCMV strains that showmonocyte-derived dendritic-cell (DC) tropism, like HCMV strain VHLE, isa candidate vector vaccine. The monocyte-derived dendritic-cell (DC)tropism depends on the propagation in according cell lines, moreparticularly the propagation on DCs following co-culture with infectedendothelial cells. Briefly, Peripheral blood mononuclear cells (PBMC)are isolated from peripheral blood from healthy donors and cultivated inRPMI supplemented with 10% fetal bovine serum (FBS) supplemented with800 U human recombinant granulocyte macrophage-colony-stimulating factorml⁻¹ (GM-CSF) and 500 U human recombinant IL-4 ml⁻¹ (R&D systems) for5-7 days, according to a reported procedure (Sallusto, F. andLanzavecchia, A., 1994, J Exp Med, 179(4):1109-18). Following thisprocedure, more than 90% adherent cells belong to the immature DCphenotype (CD1a⁺, HLA-DR^(low), CD14⁻, CD83⁻, CD86^(low), HLA-ABC⁺,CD40⁺). DCs, following 24 h incubation with cell-free HCMV preparations,obtained after virus adaptation to growth in HUVEC, were co-culturedovernight with autologous PBMCs. Co-culture medium is RPMI-10% FBSsupplemented with 10 μg brefeldin A ml⁻¹ (Sigma) to prevent release ofcytokines. Cell cultures are incubated overnight at 37° C. in 5% CO₂atmosphere. Ability of viral DCs infection is confirmed by FACS analysis(DC phenotype, detection of IE-1, pp65 or gB) and by determination ofviral titer.

It will be also acknowledged by a person skilled in the art thatcross-presentation is considered to play an important role in the immuneresoponse against beta-herpesvirus infection as well as in the immuneresponse against various other pathogens. In an embodiment thebeta-herpesvirus of the present invention is capable of infectingfibroblasts. In an embodiment the beta-herpesvirus of the presentinvention is capable of infecting fibroblasts only or with highpreference. In an embodiment the beta-herpesvirus of the presentinvention is capable of infecting parenchymal cells, comprisinghepatocytes and smooth muscle cells, preferably smooth muscle cells ofthe gastrointestinal tract (Sinzger, C. 1996, supra). In an embodimentthe beta-herpesvirus of the present invention is capable of infectingparenchymal cells only or with high preference. Examples ofbeta-herpesviruses having a tropism limited to certain cell types whichcan be used as a basis for the beta-herpees virus according to thepresent invention were described in Revello and Gerna (Revello, M. G.,Gerna, G., 2010, Rev Med Virol, 20(3):136-55). In a further embodimentof the beta-herpesviurs according to the present invention, thebeta-herpesvirus persists in the subject infected with saidbeta-herpesvirus of the present invention. In a further embodiment ofthe beta-herpesvirus according to the present invention, thebeta-herpesvirus does not persist in the subject infected with saidbeta-herpesvirus of the present invention. In an embodiment wherein thebeta-herpesvirus does not persists in the subject infected with saidbeta-herpesvirus, the genome of the beta-herpesvirus is lost over time.

In embodiments of the beta-herpesvirus of the present invention thebeta-herpesvirus preferably is a CMV, more preferably an HCMV. In anembodiment of the beta-herpesvirus of the present invention thebeta-herpesvirus is an HCMV, and/or an HCMV wherein the HCMV is derivedfrom a Bacterial Artificial Chromosome. The HCMV and/or the BacterialArtificial Chromosome can be derived from different HCMV strains. Inconnection therewith it is a preferred embodiment where the HCMV of thepresent invention has the ability to infect a wide range of differentcell types, basically all cell types that will be infected by a clinicalisolate of CMV and HCMV in particular. In a further embodiment the HCMVof the present invention has a tropism like a wild type beta-herpesvirusstrain. In a still preferred embodiment the HCMV of the presentinvention is capable of infecting antigen presenting cells, includingbut not limited to dendritic cells and macrophages. In a furtherpreferred embodiment the HCMV of the present invention is capable ofinfecting professional antigen presenting cells only. In a furtherpreferred embodiment the HCMV of the present invention is capable ofinfecting fibroblasts. In a still further preferred embodiment the HCMVof the present invention is attenuated. It will be understood by aperson skilled in the art that a virus is preferably attenuated todifferent degree. In an embodiment of the HMCV of the present inventionthe attenuation of the HCMV is preferably due to expression of acellular ligand for immune receptors, more preferably due to expressionof a cellular ligand for an NK cell receptor. In a still furtherpreferred embodiment the HCMV of the present invention is attenuated dueto deficiency in at least one gene product encoded by an immunemodulatory gene. In a still further preferred embodiment the HCMV of thepresent invention is attenuated due to the deletion of at least oneessential viral gene.

In an embodiment of the beta-herpesvirus according to the presentinvention the attenuation of the beta-herpesvirus of the presentinvention is due to expression of a cellular ligand, more preferably dueto expression of a cellular ligand for an NK cell receptor and/or due todeficiency in at least one gene product encoded by an immune modulatorygene and/or due to the deletion of at least one essential viral gene.

In an embodiment of the beta-herpesvirus according to the presentinvention the beta-herpesvirus is derived from a Bacterial ArtificialChromosome. A “bacterial artificial chromosome” also referred to hereinas BAC, as used herein preferably is a DNA construct, based on afunctional fertility plasmid (or F-plasmid), used for transforming andcloning in bacteria, usually E. coli. A person skilled in the art willbe aware that various herpesviruses were cloned as a bacterialartificial chromosome as for example described in Messerle, M. et al.,(Messerle M. et al., 1997, Proc Natl Acad Sci USA,94(26):14759-63). Aperson skilled in the art will also be aware of various techniques whichallow for mutagenesis of said herpesvirus cloned as a BAC such as forexample described by Tischer et al. (Tischer, B. K. et al., 2006,Biotechniques, 40(2):191-7). Transfection of the BAC plasmid containingthe beta-herpesvirus genome into respective eukaryotic cells, such astransfection of HCMV BAC into HFF, leads to a productive virusinfection. As has been outlined above the HCMV of the present inventionand/or the HCMV of the present invention derived from a BacterialArtificial Chromosome can be derived from different HCMV strains.

In connection with the embodiments of the HCMV of the present inventionthe HCMV of the present invention and/or the HCMV of the presentinvention derived from a Bacterial Artificial Chromosome are preferablyderived from different HCMV strains and/or Bacterial ArtificialChromosomes derived from respective HCMV strains comprising AD169 BAC(Human cytomegalovirus strain AD169, complete genome, 229,354 bp linearDNA, Accession: X17403.1, GenBank: GI: 59591), modified AD169-BAC whichis modified according to the modifications described in Borst et al.(Borst, E. M. et al., 1999, J Virol, 73(10):8320-9), TB40E BA (Humanherpesvirus 5 strain TB40/E clone TB40-BAC4, complete sequence, 229,050bp linear DNA, Accession: EF999921.1, GenBank: GI:157779983), 229,700 bplinear DNA, Accession: AC146904.1, Genebank GI: 37777313), HumanHerpesvirus 5 Toledo-BAC isolate (complete sequence, 226,889 bp linearDNA, Accession: AC146905.1, Genebank GI: 37777314), Human Herpesvirus 5TR-BAC isolate (complete sequence, 234,881 bp linear DNA,Accession:AC146906.1, Genebank GI:37777315), Human Herpesvirus FIX-BACisolate (complete sequence, 229,209 bp linear DNA, Accession:AC146907.1, Genebank GI:37777316), Human herpesvirus 5 transgenic strainMerlin (complete genome, 243,724 bp circular DNA, Accession:GU179001.1,Genebank GI:270311373) and Towne strain. In connection therewith it hasto be noted that the FIX BAC clone (Hahn, G. et al., 2002, J Virol,76:9551-5) was prepared from the VR1814 clinical isolate (Revello, M. etal., 2001, J Gen Virol, 83:1993-2000) by substituting BAC sequences forthe IRS1-US6 region using the method of Borst et al. (Borst, E. M. etal, 1999, supra). In connection therewith it will be acknkowledged thatRV-FIX and RV-FIX deletion mutants miss single genes outside theUL131-128 locus. More particularly, RV-FIX misses UL45, UL127 and UL132.HCMV revertants, such as AD169 rev and Towne rev, reacquire bothEC-tropism and leukocyte transmissibility after loss of both propertiesin HELF (Gerna, G. et al., 2002, supra). Furthermore, Merlin strain hasbeen described by Stanton et al., (Stanton, R. J. et al., 2010, J ClinInvest, 1; 120(9):3191-208), BAC cloned AD169 has been described byBorst et al. (Borst, E. M. et al., 1999, supra), Full-length AD169 hasbeen described by Hobom et al., (Hobom, U. et al., 2000, JVirol,74(17):7720-9), BAC Cloning Towne and Toledo strains has beendescribed by Hahn et al., (Hahn, G. et al., 2003, Virology,307(1):164-77), BAC cloning TB40E strain has been described by Sinzgeret al., (Sinzger, C. J. et al., 2008, supra) HCMV strain TRhas beendescribed by Murphy et al., (Murphy, E. et al., 2003, Proc Natl Acad SciUSA. 100:14976-14981), Towne BAC has been described by Marchini et al.(Marchini, A. et al., 2001, J Virol,75(4):1870-8), Construction of aself-excisable bacterial artificial chromosome containing the humancytomegalovirus genome and mutagenesis of the diploid TRL/IRL13 gene hasbeen described by Yu et al., (Yu, D. et al., 2002, JVirol,76(5):2316-28).

So as to determine whether the immune response elicited by thebeta-herpesvirus of the present invention and particularly the humancytomegalovirus of the present invention comprises at least neutralizingantibody a person skilled in the art will know assays to determine thetiter and/or how to determine the neutralizing capacity of saidneutralizing antibodies such as the assay described by Cui et al. (Cui,X. et al., 2008, Vaccine, 26(45):5760-6).

In an embodiment of the beta-herpesvirus of the present invention thebeta-herpesvirus is deficient in at least one gene product encoded by anessential gene. It will be acknowledged by a person skilled in the artthat deficiency in at least one gene product encoded by an essentialgene will result in a virus which is not replicating in vitro and/or invivo and/or is not able to produce progeny in vitro and/or in vivo. Suchvirus can be produced by trans-complementing the deficient gene product(Mohr, C. A. et al., 2010, supra). In an embodiment of thebeta-herpesvirus of the present invention deficient in a gene productthe beta-herpesvirus is preferably spread-deficient. Spread-deficient asused herein, preferably means that the virus which is spread-deficientinfects a cell and no viral particle is released from the infected cell,whereby the viral DNA is replicated, the viral proteins except thosewhich are deleted in accordance with the present invention are expressedin the infected cell, preferably all viral glycoproteins are expressed,more preferably all viral glycoproteins are expressed, that mediateentry of the virus into a cell, whereby, preferably, the cell is anendothelial and/or an epithelial cell.

As used herein, the term “deficient in at least one gene product”preferably means that the at least one gene product which is abiochemical material such as a nucleic acid, DNA, RNA or a peptide,polypeptide or protein, resulting from expression of the gene does notshow at least one of the functions displayed by said gene product in thewild type strain. Preferably, all of the functions of said gene productin the wild type strain are not shown. This is preferably the result ofa complete or partial deletion or mutation of the gene coding for saidgene product, of a complete or partial deletion or mutation of thenucleic acid controlling the expression of the gene coding of said geneproduct, of a truncation of said gene product, of the inhibition of theotherwise complete gene product, or of the at least partial or completedeletion or mutation of a promoter or other elements necessary for thetranscription of said gene. For example, a beta-herpesvirus of thepresent invention deficient in a gene product of an immune modulatorygene such as US11 will improve antigen presentation in the HLA class Ipathway.

The term “foreign antigen” and the term “heterologous antigen” as usedherein preferably mean an antigen which is constituted by a peptide,oligopeptide, polypeptide or protein encoded by a heterologous nucleicacid. In connection therewith a nucleic acid coding for an antigen andepitope, respectively, of a pathogen is also referred to herein asadditional heterologous nucleic acid, whereby such heterologous nucleicacid is preferably one which is not part of a wild type genome of abeta-herpesvirus.

In an embodiment the use of beta-herpesvirus of the present inventionfor the manufacture of a medicament, the medicament is for the treatmentand/or prevention of a disease or condition associated withbeta-herpesvirus infection, preferably human cytomegalovirus infection.

In connection therewith it will be understood that human cytomegalovirus(HCMV) is ubiquitous in human populations and establishes lifelonginfection. In subjects with normal immune systems, HCMV does typicallynot cause serious disease. However, in immunosuppressed patients, forexample AIDS and transplant patients, and immunologically immaturenewborns, HCMV can cause grave disease and even death. Congenital HCMVinfection, i.e. HCMV infection present at birth, is the most commonviral infection that is transmitted in-utero to the human fetus andoccurs in about 0.4%-1.2% of live borne infants in most regions of theworld. Symptomatic infections following congenital HCMV infection occurin about 10% of infected infants. Symptoms include clinicalmanifestations that include but are not limited to, (1) microcephaly orother neurologic symptoms attributable to HCMV associated neurologicdisease (2) hepatosplenomegaly with jaundice, and (3) thrombocytopeniawith petechial/purpuric rashes.

In an embodiment of the beta-herpesvirus of the present invention thebeta-herpesvirus is for use in the manufacture of a vaccine, wherein thevaccine is or is suitable for the administration to a donor of atransplant and/or to a recipient of a transplant. It is within thepresent invention that the vaccine and/or the beta-herpesvirus of thepresent invention is administered to a subject which is scheduled asrecipient or donor for singular transplantation, e.g. of a solid organ,cells, or bone marrow, or a subject which is scheduled as recipient ordonor for transplantation, preferably repeated transplantation, of e.g.,cells or bone marrow. It is also possible that the vaccine and/or thebeta-herpesvirus of the present invention is administered to a subjectsuspected to be or willing to be a donor or is suspected to be arecipient of a transplantation in the future.

In connection therewith the beta-herpesvirus of the present invention ispreferably administered to the donor or recipient prior totransplantation or planned transplantation or as a preventive measure.Accordingly, in an embodiment the donor is a potential donor and/or therecipient is a potential recipient.

In another aspect of the present invention the beta-herpesvirus of thepresent invention is part of a pharmaceutical composition. Preferably,such pharmaceutical composition contains, apart from thebeta-herpesvirus of the present invention and/or a nucleic acid codingfor the same, a pharmaceutically acceptable carrier. The ingredients ofsuch pharmaceutical composition and their respective amount contained insuch pharmaceutical composition are either known to a person skilled inthe art or can be determined by routine measures. It will be furtheracknowledged by a person skilled in the art that such pharmaceuticalcomposition is for or is for use in the treatment of the diseases andconditions as disclosed herein in connection with the beta-herpesvirusof the present invention and its use.

It will be acknowledged by a person skilled in the art that as far asthe experimental evidence provided in the example part of the instantapplication is based on MCMV, such evidence can be directly andimmediately transferred to HCMV, so that the claimed invention isplausible to a person skilled in the art also for this reason. Thereason is that the genomes of different herpesvirus strains includingHCMV and MCMV are linearly correlated and the mode of action of HCMV ina human host and the mode of action of MCMV in a murine host areessentially identical.

The various SEQ ID NOs., the chemical nature of the nucleic acidmolecules and peptides according to the present invention, the actualsequence thereof and the internal reference number are summarized in thefollowing table.

SEQ. ID. NO: Sequence internal reference  1 YPHFMPTNLIE1/m123 (₁₆₈YPHFMPTNL₁₇₆) peptide  2 AGPPRYSRIm164 (₁₆₇AGPPRYSRI₁₇₅) peptide  3 GYKDGNEYI List (₉₁GYKDGNEYI₉₉) peptide 4 5′- primer for amplification of the RAE-1γgcacccgacgatctgacattgtccagtgtgccexpression cassette plus kanR, wherein italic ggtcgcacgaacatccctagttattaatagtaat and underlined letters are homologous to nt c-3′210196 to 210242 of the genome of mousecytomegalovirus according to Genbankaccession number: U68299.1, flanking m152  5 5′-primer for amplification of the RAE-1γ tgtcaccgctccacgtttcaccgtcggtctcccexpression cassette plus kanR, wherein gatcgctagcctgtaca caggaacacttaacgitalics italic and underlined letters are gctga-3′homologous to nt 211427 to 211378 (lower strand) of the genome of mousecytomegalovirus according to Genbankaccession number: U68299.1, flanking m152.  6 5′-primer List swap fw_1; wherein lower case gactactgtcggacgtggggcgctgacaatat letters represent homology region to m164 attcatttccatctttgtaaccAGGATG ORF in the MCMV genome, lowercase letters ACGACGATAAGTAGGG-3′in bold and underlined represent the regioncoding for peptide ₉₁GYKDGNEYI₉₉ - List(SEQ. ID. NO: 3) and capital bold lettersrepresent homology to Tischer kanamycinresistence cassette (Tischer, B. K.  et al, 2006, supra.) . . .  7 5′-primer List swap fw_2; gatcgagccggtggtaccggacgcggcggagccgttcggaaaggactactgtcggacgtggg gcgctgac-3′  8 5′-primer List swap rv_1; wherein lower caseggttacaaagatggaaatgaatatattgtcaletters represent homology region to m164 gcgccccacgtccgacagtagtcCAACCORF in the MCMV genome, lowercase letters AATTAACCAATTCTGATTAin bold and underlined represent the region G -3′coding for peptide ₉₁GYKDGNEYI₉₉ - List(SEQ. ID. NO: 3) and capital bold lettersrepresent homology to Tischer kanamycinresistence cassette (Tischer, B. K.  et al, 2006, supra) . . .  9 5′-primer List swap rv_2 atggcctggttgttgacggcccagaagatgcgcgagtaccgaggagggcccgcggttacaaag atggaaatgaatatatt- 3′ 10 SIINFEKLSIINFEKL-peptide 11 5′- primer PR8HA fw GCCGCCATGAAGGCAAACCT ACTGG-3′ 125′- primer PR8 V5 rv CGTAGAATCGAGACCGAGG AGAGGGTTAGGGATAGGCTTACCGATGCATATTCTGCACT GCAAAGATCC-3′ 13 5′- primer PR8 SIINFEKLTCACAGTTTTTCAAAGTTGA TTATACTCGTAGAATCGAGA CCGAGGAGAGGGTTAGG-3′ 14 5′-primer Headless fw GGAGGCAACACGAAGTGTC AAACACC-3′ 15 5′-primer Headless rv GCCACCACATAGTTTTCCGT TGTGGC-3′ 16 GAPINSATAMMycobacterium tuberculosis H-2Db immunodominat epitope 309-GAPINSATAM-318 17 5′- primer m164 GAP fw; lower case letterscgcccgctgccacgatggcctggttgttgacgrepresent homology region to m164 ORF in gcccagaa catggcggtggccgagttgatcMCMV genome, underlined letters in bold ggggcgcc gtcagcgcccca GCCAGTrepresent homology regions between primers, GTTACAACCAATTAACC-3′)italic letters are I-SceI restriction sitesequence and capital letters representhomology to Tischer kanamycin resistencecassette (Tischer, B. K. et al., 2006, supra) 18 5′-primer m164 GAP rv; lower case letters gccgttcggaaaggactactgtcggacg

represent homology region to m164 ORF in

ggcgccccgatcaactcggcca MCMV genome, underlined letters in bold ccgccatgTAGGGATAACAGGG represent homology regions between primers, TAATCGAT-3′italic letters are I-SceI restriction sitesequence and capital letters representhomology to Tischer kanamycin resistancecassette (Tischer, B. K. et al, 2006, supra). 19 5′-primer to amplify ULBP2 ORF; wherein the GTCGGTACCGTCGCAGTCT-underlined letters indicate homology to nt TCGGTCTGACCACCGTAGAAposition 182904 - nt position 182857 (lower CGCAGAGCTccaccATGGCAGstrand) of the mouse cytomegalovirus CAGCCgccGCTACC-3′sequence according to GenBank Accession No.: U68299.1; 20 5′-primer to amplify ULBP2 ORF; wherein the cccGGATCCctctcc TCA GATGCStop codon (in inverse orientation) is CAGGGAGGATGAAG-3′indicated in bold and underlined. 21 5′-primer to amplify insert including ULBP2 GACACCGGGCTCCATGCTGAORF and including the promoter and KanR; CGTAGGTACCGACTGGGGTCwherein the capitalized letters are AAAAGCCTttaaacggtactttcccataghomologous to nucleotides 55134-55181 of c-3′the TB40E BAC (Genbank: EF999921.1) 22 5′-primer to amplify insert including ULBP2 CTTATAGCAGCGTGAACGTTORF and including the promoter and KanR; GCACGTGGCCTTTGCGGTTAwherein the capitalized letters are TCCGTTCAGgaacacttaacggctga-homologous to nucleotides 55963-55915 3′(lower strand) of the TB40E BAC (Genbank: EF999921.1; Sbjct 55963CTTATAGCAGCGTGAACGTTGCACGTG GCCTTTGCGGTTATCCGTTCAG 55915)and lower case letters are homologous tosequences on the plasmid carrying the MCMV MIEP, the ULBP2 ORF and KanR23 5′- sequencing primer GGCGATGCGGTATCGCGCAC A-3′ 24 5′-sequencing primer GACACCTGTTCGTCCAGAAT C-3′ 25 5′- Primer ie4fwdTGACTTAAACTCCCCAGGCA A-3′ 26 5′- Primer ie4rev TAGGTGAGGCCATAGTGGCA G-3′27 5′- Primer glralfwd TGCCTGTTCTTTGCAGTCTGT- 3′ 28 5′- Primer glralrevAGTCGAGTGAAGGGTAACG AGC-3′ 29 RALEYKNL IE3 (⁴¹⁶RALEYKNL⁴²³) peptide 30TVYGFCLL m139 (⁴¹⁹TVYGFCLL⁴²⁶)peptide 31 HGIRNASFIM45 (⁹⁸⁵HGIRNASFI⁹⁹³)peptide 32 SSPPMFRV M38 (³¹⁶SSPPMFRV³²³)peptide 33GTGTATGTGGCCCGACGGGC primer m152fw GG 34 CGCGGGCTACTCCCGAAAGAprimer m152rv GTAACATC 35 ATGGCCAAGGCAGCAGTGAC primer RAEfw 36TGCTCGACCTGAGGTAATTA  primer RAErv TAACCC 37 N-IYSTVASSL-CH-2Kd-Balb/c restricted HA peptide HA533-541 (N-IYSTVASSL-C) immunodominant epitope 38 YPYDVPDYAH2b-B6 mice restricted peptide HA114-122(YPYDVPDYA)- this one is erased in case of headless mutatnt 39TATATAGACTGAAGCGGAGT 20 nt homologous to sequences immediatelydownstream of the 3′-end of the UL11 ORF of the CMV strain TB40E 40CAGCTTTTGAGTCTAGACAG UL11-HA-fw GGGAACAGCCTTCCCTTGTA AGACAGAATGaagg-caaacctactggtcc 41 GAGTCGTTTCCGAGCGACTC UL11-HA-rev GAGATGCACTCCGCTTCAGTCTATATATCA 42 AAGUGACGGUGAGAUCCAG hcmv-miR-UL112 GCU(MIMAT0001577(MirBase) (Stern-Ginossaret al., 2008, supra, Stern-Ginossar  et al., 2007, supra) 43 TIHDIILECVE6 29-38 (TIHDIILECV; restricted by the HLA-A0201 molecule), 44TIHDIILEC E6 29-37 (TIHDIILEC; restricted by B48), 45 HDIILECVE6 31-38 (HDIILECV; restricted by B4002) 46 FAFRDLCIVYE6 52-61 (FAFRDLCIVY; restricted by B57),

It will be acknowledged by a person skilled in the art and it is insofaralso within the scope of the present invention that each and any of theabove nucleic acid sequences can be replaced by a nucleic acid sequencewhich, due to the degeneracy of the genetic code, code for the same orfunctionally homolog peptide, polypeptide and protein, respectively, asthe above indicated nucleic acid sequences.

The present invention is now further illustrated by the followingfigures and examples from which further features, embodiments andadvantages of the present invention may be taken.

EXAMPLES Example 1 Materials and Methods General Laboratory Reagents andEquipment

Materials and reagents which used in the present application are listedin Table 1.The laboratory equipment used in the present application is listed inTable 2.

TABLE 1 Materials and reagents. REAGENTS DISTRUBUTOR NAME αB220 clonePA3-6B2 eBioscience αCD3 clone SP7 Abcam αCD3 clone 145-2C11 eBioscienceαCD8 clone 53-6.7 eBioscience αCD11c clone N418 eBioscience αCD11b cloneM1/70 eBioscience αCD27 clone LG.7F9 eBioscience αCD44 clone IM7eBioscience αCD62L clone MEL-14 eBioscience αCD69 clone H1.2F3eBioscience αCD127 clone A7R34 eBioscience αIFNg cloneXMG1.2 eBioscienceαKLRGclone 1 2F1 eBioscience αMHCII clone M5/114.15.2 eBioscience αNKp46clone 29A1.4 eBioscience αPD-1 clone J43 eBioscience αTNFa cloneMP6-XT22 eBioscience Acetic acid (glacial) BDH AEC staining kitSigma-Aldrich Co. Agarose Carl Roth Amminium chloride Kemika AmpicillinEMD Chemicals Inc. Arabinose Carl Roth Aquatex Merck 2-mercaptoethanolInvitrogen - Gibco Cell Culture Systems Bacto Agar Carl Roth Biotinblocking system Dako Brain heart infusion (BHI) broth Difco LaboratoriesBoric acid Carl Roth Brefeldin A eBioscience Bovine serum albumine (BSA)Carl Roth Chloramphenicol EMD Chemicals Inc. Chloroform EMD ChemicalsInc Defibrinated sheep blood Biognost d.o.o. DMEM (Dulbecco's ModifiedEagle Pan Biotech GmbH Medium) DMSO Sigma-Aldrich Co. dNTPs Hoffmann-LaRoche Ltd EDTA Carl Roth Eosin Thermo Scientific Ethanol T.T.T. d.o.o.Ethidium bromide BDH Foetal Bovine Serum Pan Biotech GmbH Gel loadingbuffer 10X Blue Juice Invitrogen Glucose Carl Roth Glycerol EMDChemicals Inc. Hematoxillin Thermo Scientific Hydrocloric acid, 37%Carlo Erba Reagents Isoamyl alcohol BDH Isopropanol EMD Chemicals Inc.Kanamycin EMD Chemicals Inc. L-Glutamin Invitrogen - Gibco Cell CultureSystems MEM 10x Invitrogen - Gibco Cell Culture Systems Methanol T.T.T.d.o.o. Methylcellulose Sigma-Aldrich Co. mi 100 bp DNA marker GO LadderMetabion mi 1 kbp DNA marker GO Ladder Metabion Nutrient agar with NaClBiolife Paraformaldehyde Sigma-Aldrich Co. Penicillin/Streptomycin PanBiotech GmbH Phenol EMD Chemicals Inc. PBS-Buffer Dulbecco (PhosphateBuffered Pan Biotech GmbH Saline) Potassium acetate Carl Roth Potassiumhydrogen carbonate Kemika QIAquick PCR purification kit Qiagen IncQIAplasmid MIDI kit Qiagen Inc Restriction enzymes New England Biolabs,Inc. RPMI Pan Biotech GmbH Saponin, from quillaja bark Sigma-Aldrich Co.Sodium azide Sigma-Aldrich Co. Sodium chloride Kemika Sodium dihydrogenphosphate 12-hydrate Kemika Sodium dodecyl sulfate BDH Sodium hydroxideKemika Streptavidin eBioscience Sucrose Carl Roth SuperFect TransfectionReagent Qiagen Inc. Tris base Carl Roth Trypan Blue Stain InvitrogenTrypsin-EDTA 10X Invitrogen - Gibco Cell Culture Systems XyleneT.T.T.d.o.o.

TABLE 2 Laboratory equipment. LABORATORY EQUIPMENT DISTRIBUTOR NAME BDFacs Aria Cell Sorter BD Biosciences Biofuge 13 microcentrifuge ThermoElectron Corporation - Heraeus Bottle Top filters TPP Techno PlasticProducts Cell culture dish Orange Scientific Centrifuge C412 JouanCentrifuge 5417R Eppendorf Centrifuge tubes Beckman CO2 Incubator 3326Heraeus DNA engine Peltier thermal cycler Bio-Rad Laboratories Ltd.Electroporation cuvettes Gene Pulser, BIORAD Eppendorf ThermomixerEppendorf Gene pulser II electroporator Bio-Rad Laboratories Ltd.Horizontal Gel Electrophoresis System Owl Separation Systems IncubatorB6 Heraeus JA-10 rotor Beckman Coulter Canada, Inc. J2-MI highspeedcentrifuge Beckman Coulter Canada, Inc. Microscope slides Carl RothMicroscope Olympus CK2 Olympus Microscope Olympus BX51 Olympus PipettesGilson Syringe filters TPP Techno Plastic Products Thermo Biomate 3spectrophotometer Thermo Electron Corporation Transilluminator UV HVDLife Sciences Water bath Kottlermann Labortechnik2.4G2 supernatant (Fc block), aCD8, aCD4 and aNKG2D antibodies wereproduced at the Center for Proteomics, Faculty of Medicine, Universityof Rijeka.Peptides IE1/m123 (₁₆₈YPHFMPTNL₁₇₆; SEQ. ID. NO: 1), also known as a pp89 derived peptide, m164 (₁₆₇AGPPRYSR₁₁₇₅; SEQ. ID. NO: 2)-peptide [and₉₁GYKDGNEYI₉₉ (SEQ. ID. NO: 3) were synthesized by JPT PeptideTechnologies, Germany. Tetramers were provided by NIH Tetramer Facility.

Buffers and Solutions 0.1M Citrate Buffer pH 5.5

Solution A: 21.01 g 0.1M citric acid in 1 L of distilled water.Solution B: 35.81 g 0.1M Na2HPO4x12H2O in 1 L of distilled water.Solutions A and were mixed in 1:1 ratio and titrated with citric acid ordisodium hydrogen phosphate solution for pH adjustment.

DNA Isolation Buffers:

Buffer I: 50 mM glucose; 10 mM EDTA; 25 mM TRIS pH 8.0

Buffer II: 0.2 M NaOH; 1% SDS;

Buffer III: 3M potassium acetate;Buffer I was autoclaved before usage. Buffers I and III were usedice-cold. The same stock solution can be used for one month if stored at4° C. Buffer II was always prepared fresh prior to mini preparation.10× Lysing solution

For IL: 89.9 g NH4Cl; 10 g KHCO3; 0.370 g EDTA

pH was adjusted to 7.3, solution was sterile filtered and stored at 2-8°C.

FACS Medium 1 L PBS; 0.1% NaN3; 1% BSA 4% PFA pH 7.2 For 1 L:

40.0 g paraformaldehyde; 5-10 g NaOH; PBS buffer PBS was heated to 60°C. in water bath. Paraformaldehyde was added and solution was mixed 1-2h on magnetic stirrer. pH was adjusted to 7.2, PBS was added up to 1 Land solution was filtered.

2% PFA pH 7.2

2% PFA pH 7.2 was prepared by 2× dilution of 4% PFA pH 7.2 in PBS.TBS buffer200 mL 1M TrisHCl pH 7.5 (121.14 g Tri sin 1 L od distilled water, addconcentrated HCl)300 mL 1M NaCl (58.44 g in 1 L distilled water add aqua up tp 2 L

Trypanblue:

4.0 g of trypanblue dye was dissolved in u 100 mL PBSa in dark bottle.After 5-7 days at +4° C. solution was filtered.

Trypsin/EDTA

Trypsin/EDTA solution diluted 1:10 with PBS was used.Final concentration: 0.5 g/l trypsin, 0.2 g/l EDTA, pH 7 0.4 to 7, 6.Virus suspension buffer (VSB)/15% (w/v) sucrose:50 mM Tris-HCl; 12 mM KCl; 5 mM Na₂EDTA; 15% sucrose

Cell Culture Media

Foetal bovine serum (FCS)FCS was before usage decomplemented by heating at 56° C. in water bathfor 1 h.

3% DMEM (Dulbecco's Modified Eagle Medium)

500 mL DMEM; 3% (v/v) FCS; 100 U/ml Penicillin; 0.1 mg/ml Streptomycin

5% RPMI

500 mL RPMI; 3% (v/v) FCS; 500 mL 2-mercaptoethanol; 100 U/mlPenicillin; 0.1 mg/ml Streptomycin

10% RPMI

500 mL RPMI; 3% (v/v) FCS; 500 mL 2-mercaptoethanol; 100 U/mlPenicillin; 0.1 mg/ml Streptomycin

Methyl Cellulose

Methyl cellulose was prepared according to manufacturer instructions.In a glass bottle, 8.8 g methyl cellulose and 0.88 g NaHCO3 was added in350 mL of distilled water, left for 10 days in the fridge to dissolve(with periodically shaking) and autoclaved afterwards at 121° C. for 20min. 40 mL of 10×MEM, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/mlstreptomycin, 5% (v/v) FCS was added while stirred on magnetic stirrer.It was stored at 4° C.

PBS-Buffer Dulbecco (Phosphate Buffered Saline): 140 mM NaCl; 2.7 mMKCl; 6.5 mM Na2PO4; 1.5 mM KH2PO4, (pH 7.4) Bacteria Culturing MediaNutrient Agar

Nutrient agar with NaCl was prepared according to manufacturerinstructions. 28 g of agar was dissolved in 1000 mL of distilled waterand autoclaved for 15 min at 121° C. In cooled agar 50 mL of blood agarwas added and plated in sterile Petri dish.

LB Media

10.0 g Bacto-Tryptone; 5.0 g Bacto-yeast extract; 10.0 g NaCl; ddH2O upto 1 L

LB Plates

10.0 g Bacto-Tryptone; 5.0 g Bacto-yeast extract; 10.0 g NaCl; 15.0 gBacto-agar; ddH2O up to ILLB media was autoclaved and cooled to 55° C. Appropriate antibioticswere added and media was plated in sterile bacteria culture plates.

Cells and Viruses.

Mouse embryonic fibroblasts, also referred to herein as MEFs, SVEC4-10(ATTCC no. CRL-2181), NIH 3T3 (ATCC CRL-1658) and B12 fibroblasts (DelVal, M. et al., 1991, Cell 66(6):1145-53), were grown as described inJonjic et al. (Jonjic, S. et al., 2008, Methods Mol Biol 415:127-149).MCMV, MW97.01, derived from a bacterial artificial chromosome, alsoreferred to herein as BAC, has previously been shown to be biologicallyequivalent to the MCMV Smith strain (VR-194 [reaccessioned as VR-1399];American Type Culture Collection) and is also referred to hereinpreferably as WT-MCMV. The recombinant strain Δm152-MCMV was generatedas described by Wagner et al. (Wagner, M. et al., 1999, J Virol73(8):7056-60; Wagner, M. et al., 2002, J Exp Med 196(6):805-16).Viruses were propagated on MEFs and concentrated by sucrose gradientultracentrifugation as described by Jonic et al. (Jonjic, S. et al.,2008, supra). Salivaary gland derived MCMV, also referred to hereinpreferably as SGV MCMV, was used as a third passage and prepared asdescribed by Jonjic et al. (Jonjic, S. et al., 2008, supra).

Cell culture techniques were performed using a sterile cabinet, as wellas sterile glass and plastic material. For the cultivation of cellsCO₂-incubators providing the following conditions, namely 37° C., 5% CO₂(v/v) and a saturated water vapor atmosphere (95% (v/v) relativehumidity), were used.

For determining cell numbers, a cell suspension was mixed with trypanblue dye and transferred into a Neubauer-counting chamber. The cellnumber was determined according to the following formula I:

CN/ml=N/n×V×10⁴  (I)

wherein CN means cell number, N means number of counted cells, n meansnumber of large squares counted, and V means dilution factor, andwherein the chamber factor is 10⁴.

Preparation of MEF

MEFs were isolated from 17 days old mouse embryos. The embryos wereremoved from a pregnant mouse and minced in a Petri dish using scissors.Tissue fragments were rinsed with PBS and transferred to a 500 mLErlenmeyer flask. After addition of 30 mL trypsin solution the flask wasstirred at 37° C. for 30 min. Additional 30 mL of trypsin solution and10 mL of PBS wad added and stirred at 37° C. for 30 min two more times.Cell suspension was filtered through gauze and centrifuged 10 min at1200 rpm and room temperature. Cells were resuspended in warm 3% DMEM,plated and grown till they reached confluency, which approximatelyoccurs within 2 to 3 days. After expansion of cells, aliquots thereofcan be frozen and stored at −80° C. or used.

Production of Electrocompetent Bacterial Cells

5 mL of LB broth medium containing 17 g/mL chloramphenicol wasinoculated with the BAC containing bacteria (E. coli DH10B) andincubated overnight at 37° C., with minimum shaking of approximately 200rpm. 4 mL of the resulting overnight culture were transferred into two 2mL microcentrifuge tubes and cells were pelleted by centrifugation at16000 g at 2° C. for 30 seconds.

To obtain highly electrocompetent cells, the procedure was performed onice with prechilled solutions and microcentrifuge tubes. The Pelletresulting from the centrifugation step was resuspended in 1 mL ofice-cold sterile ddH₂O by gentle pipetting. Cells were centrifuged at16000 g at 2° C. for 30 seconds and the resulting pellet was resuspendedin 1 mL of ice-cold sterile ddH₂O. Cells were centrifuged at 16000 g at2° C. for 30 seconds and the resulting pellet was resuspended in 500 μLof ice-cold sterile 10% glycerol (v/v). Cells were pooled in onemicrocentrifuge tube and centrifugation was carried at 16000 g for 60seconds at 2° C. The resulting supernatant was discarded using a pipetteand the pellet containing the bacterial cells was resuspended in 100 μLof ice-cold sterile 10% glycerol (v/v). Aliquots were snap-frozen inliquid nitrogen. Cells were stored as 50 μL aliquotes at −80° C.

Electrotransformation

E. coli DH10B cells were electroporated with the respective MCMVBAC-plasmid to introduce the plasmid pKD46, which encodes therecombination enzymes, and to excise kanamycin cassette from the MCMVBAC-genome by the electroporation of pCP20 (Borst E M et al., CurrProtoc Immunol. 2007 May; Chapter 10: Unit 10.32).

An aliquot of electrocompetent bacteria was thawed on ice and 5 ng ofplasmids and approximately 300 ng of PCR product was added.Electroporation was performed in pre-cooled electroporation cuvettes at2.5 kV, 200Ω and 25 μF. Subsequent to the electroporation step, 500 μlLB-medium was added to the bacteria and the such obtained culture wasincubated for 1 h at 300 rpm in a thermo-shaker at 30° C. afterelectroporation with pCP20, or 37° C. after electroporation with pKD46or PCR product, respectively. After incubation, bacterial cells wereplated on LB agar plates containing appropriate antibiotics, accordingto selection marker present in the plasmids or PCR products andincubated at 30° C. over night.

Induction of Expression of Red Genes from pKD46

After electroporation of pKD46 plasmid into bacterial cells containingthe respective MCMV-BAC, a single colony was inoculated in 5 mL of LBmedium containing 17 μg/mL chloramphenicol and 50 μg/mL ampicillin andsuch obtained culture was incubated at 30° C. and 200 rpm overnight.Such obtained overnight culture was than diluted 1:40 into 100 mL of LBmedium containing 17 μg/mL chloramphenicol and 50 μg/mL ampicillin andincubated at 30° C. and 200 rpm for 3 h, or until OD_(600nm) reached0.5-0.6. Expression of recombination enzymes was induced by adding 1 mLof freshly prepared 10% arabinose solution (w/v) (final concentration0.1%) and incubation for 1 h at 30° C. and 200 rpm. Cells were then madeelectrocompetent as described herein.

Isolation of Viral DNA from Viral Particles

To isolate DNA from MCMV particles, the supernatant of infected mouseembryonic fibroblasts was used. MEFs were grown in 3% DMEM in 10-cm cellculture dish to 90% confluence and were infected with the supernatantsfrom transfected cells at multiplicity of infection (MOI) of 0.1.Incubation was carried at 37° C. and 5% CO₂ until complete cytopathiceffect was observed, usually 5 to 6 days post infection. Supernatantswere harvested and virions released in the supernatants were pelleted byultracentrifugation for 1 h at 100 000 g at 4° C. Pellets werere-suspended in 500 μL of 50 mM TRIS-HCl pH 8.0/1 mM MgCl₂/100 μg/mLBSA, 100 U of Benzonase was added and incubated for 1 h. To inactivateBenzonase, 20 μL of 0.5 M EDTA was added. Virions were lysed by additionof 500 μL of 1% SDS. Capsid proteins were digested by 20 μL ofproteinase K (500 ng/mL). Following 3 h of incubation at 56° C., theviral DNA was purified by phenol/chloroform extraction. One volume ofphenol/chlorophorm was added to sample containing viral DNA andcentrifugation for 5 minutes at 16000 g was performed. The resultingupper aqueous phase containing viral DNA was transferred to a new tubeusing pipettes with pipette tips with cut-off ends. To assure purity ofviral DNA, 1 μL of glycogen solution (35 μg/μL) and 1/10 volume of 3 Msodium acetate pH 5.2 was added. DNA was precipitated with 0.7 volume ofisopropanol. DNA was pelleted with centrifugation (30 minutes, 16000 g,4° C.). Supernatant was discarded and pellet washed in 1 mL of 70%ethanol. Pellet was than air-dried and dissolved in 100 μL TE buffer for2 h at 37° C. Viral DNA was analyzed by restriction analysis.

Long Mini-Preparation of BAC::MCMV DNA

10 mL of LB medium with appropriate antibiotic was inoculated with asingle bacterial colony. For verification of right clone constructs, forevery mutant 10 positive clones were analyzed. Bacterial cultures weregrown for 18 h at 37° C. at 200 rpm. 500 μL of overnight culture wasstored at 4° C. for potential large scale preparation(midi-preparation). The rest of the culture was centrifuged at 4000 rpmat 4° C. for 10 minutes. Pellet was re-suspended in 2001 of ice-coldbuffer I. The alkaline lysis was accomplished by adding 300 μl of bufferII. After addition of buffer II sample was mixed gently by inverting thetube and then was incubated for 5 min at room temperature. Afteraddition of 300 μl ice-cold buffer III, followed by incubation for 10min on ice were SDS, chromosomal DNA and protein componentsprecipitated. The precipitated components were in the lower phase aftercentrifugation at 13200 rpm (16,000×g) and 4° C. for 5 minutes.Supernatant containing DNA was transferred into a new tube and DNA wasprecipitated with 0.7 volume of isopropanol. Sample was centrifuged at13200 rpm (16,000×g) and 4° C. for 30 minutes. Pellet was washed with800 μL of 70% ethanol, air-dried and dissolved in the 100 μL of TEbuffer that contained RNase (50 ng/ml) for 30 minutes at 37° C. atminimum shaking (300 rpm) in a thermomixer. The DNA was analyzed byrestriction analysis and stored at 4° C.

Construction of Recombinant Plasmids and Recombinant Viruses.

To generate MCMV expressing RAE-1γ, i.e. RAE-1γMCMV, an ORF encodingFLAG-tagged RAE-1γ was first cloned into the plasmid pGL3 (Invitrogen)together with a kanamycin resistance gene (kanR), which was insertedfurther downstream. Then, the RAE-1γ expression cassette and kanR werePCR amplified using the primers5′-gcacccgacgatctgacattgtccagtgtgccggtcgcacgaacatccctagttattaatagtaatc-3′(SEQ. ID. NO: 4) and5′-tgtcaccgctccacgtttcaccgtcggtctcccgatcgctagcctgtacacaggaacacttaacggctga-3′(SEQ. ID. NO: 5), which contained 50 nucleotides at their 5′-endshomologous to the intended integration site in the BAC-cloned MCMVgenome. The PCR fragment was integrated into the BAC by redα, -β, -γmediated recombination as described by. Borst et al, 2007 (Borst E M etal., Curr Protoc Immunol. 2007 May; Chapter 10: Unit 10.32.), thereby inaccordance with homology chosen for the intended integration sitereplacing the m152 ORF. The kanR cassette was subsequently excised withFLP recombinase (Borst et al., 2007, supra). The resulting MCMV BAC wascharacterized by restriction analysis and virus RAE-1γMCMV wasreconstituted by transfection of the BAC DNA into MEF.

Construction of MCMVList and RAE-1γMCMVList

The Dd-restricted antigenic m164 peptide ₁₆₇AGPPRYSRI₁₇₅ (SEQ.ID.NO: 2)of the genome of MCMV strain RAE-1γMCMV and of WT-MCMV was replaced withthe Kd-restricted listeriolysin O (LLO)-derived peptide ₉₁GYKDGNEYI₉₉,also referred to herein preferably as List (SEQ.ID.NO: 3), by using theshuttle plasmid pST76K-m164_List as described by Lemmermann et al.(Lemmermann et al., 2010, J Virol. 84(3):1221-36).

The primers used were List swap fw_(—)1: 5′-gacta ctgtc ggacg tggggcgctg acaat atatt cattt ccatc tttgt aaccA GGATG ACGAC GATAA GTAGG G-3′(SEQ.ID.NO: 6), List swap fw_(—)2: 5′-gatcg agccg gtggt accgg acgcggcgga gccgt tcgga aagga ctact gtcgg acgtg gggcg ctgac-3′ (SEQ.ID.NO: 7),List swap rv_(—)1: 5′-Ggtta caaag atgga aatga atata ttgtc agcgc cccacgtccg acagt agtcC AACCA ATTAA CCAAT TCTGA TTAG-3′ (SEQ.ID.NO: 8) andList swap rv_(—)2: 5′-atggc ctggt tgttg acggc ccaga agatg cgcga gtaccgagga gggcc cgcgg ttaca aagat ggaaa tgaat atatt-3′ (SEQ.ID.NO: 9),wherein lower case letters represent homology region to m164 ORF in theMCMV genome, lowercase letters in bold and underlined represent theregion coding for peptide ₉₁GYKDGNEYI₉₉-List (SEQ. ID. NO: 3) andcapital bold letters represent homology to Tischer kanamycin resistancecassette (Tischer, B. K. et al, 2006, supra). PCR was performed with thefollowing cycler conditions: An initial step for 5 min at 95° C. foractivation of Phusion HighFidelity DNA polymerase (New England BioLabs)was followed by 30 cycles of 45 s at 94° C., 60 s at 65° C., and 60 s at72° C.

Construction of the RAE-1γMCMVm164SIINFEKL-Strain.

The SIINFEKL (SEQ.ID.NO: 10) coding DNA sequence was inserted into ORFm164 of the genome of RAE-1γMCMV, which replaced the DNA sequences forthe immunodominant intrinsic m164 peptide 167-AGPPRYSRI-175 (SEQ.ID.NO:2), by using the shuttle plasmid pST76K-m164_SIINFEKL (SEQ.ID.NO:10)-(Lemmermann et al., 2010, supra) and a recA-mediated recombinationtechnique (Borst E. M. et al., 2007, supra) as described in Lemmermannet al. (Lemmermann et al., 2010, supra). Correct insertion was verifiedby restriction analysis using ApoI and sequencing.

Construction of SIINFEKL-Peptide Expressing Recombinant Viruses.

MCMV-SIINFEKL and RAE-1γMCMVSIINFEKL were constructed by orthotopicpeptide swap on the WT-MCMV or RAE-1γMCMV backbone, respectively, asdescribed previously (Lemmermann, N. A., K. Gergely, et al. (2010).“Immune evasion proteins of murine cytomegalovirus preferentially affectcell surface display of recently generated peptide presentationcomplexes.” J Virol 84(3): 1221-36). Lemmermann et al., 2010, supra

Construction of Recombinant Plasmids Containing HA Expression Cassette

Plasmids m157 pGL3 HMIEP PR8-HA full Tischer kanamycin and m157 pGL3HMIEP PR8-HA headless Tischer kanamycin were constructed to replace them157 ORF in the wild type MCMV-BAC and in Δm152-RAE1γMCMV-BAC,respectively. PR8-HA was obtained by PCR from pUC18 containing PR8-HA(UniProt P03452) as a template DNA, provided by Peter Stäheli, TheUniversity Medical Center Freiburg, Germany. The primers were PR8HA fw:5′-GCCGCCATGAAGGCAAACCTACTGG-3′ (SEQ.ID.NO: 11), PR8 V5 rv:5′-CGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCGATGCATATTCTGCACTGCAAAGATCC-3′ (SEQ.ID.NO: 12), wherein the introduced V5 tag isindicated in bold and PR8 SIINFEKL:5′-TCACAGTTTTTCAAAGTTGATTATACTCGTAGAATCGAGACCGAGGAGAGGGTTA GG-3′(SEQ.ID.NO: 13), wherein the introduced SIINFEKL tag is indicated inbold; for the amplification of the PR8 HA full form primers Headless fw:5′-GGAGGCAACACGAAGTGTCAAACACC-3′ (SEQ.ID.NO: 14) and Headless rv:5′-GCCACCACATAGTTTTCCGTTGTGGC-3′ (SEQ.ID.NO: 15) were used for theamplification of PR8 HA headless.

PCR was performed with the following cycler conditions: An initial stepfor 2 min at 98° C. for activation of HighFidelity Phusion DNApolymerase (New England BioLabs) was followed by 30 cycles of 10 s at98° C., 10 s at 60° C., and 60 s at 72° C. PCR amplified HA full ORF wascloned into pGL3 plasmid (Invitrogen) together with a Tischer-modifiedkanamycin resistance gene, also referred to herein as kan^(R) or kanR,which was inserted further downstream (see also FIG. 24). Restrictionwith BglII of plasmids m157 pGL3 HMIEP PR8-HA full Tischer kanamycin andm157 pGL3 HMIEP PR8-HA headless Tischer kanamycin resulted in obtainingthe 4.8 kbps long insert needed for subsequent mutagenesis, which ism157 flanked PR8-HA full or headless under control of HCMV immediateearly promoter, also referred to herein as HMIEP, along with theselection marker—kanamycin cassette flanked by duplicated sequences andan adjoining I-SceI restriction site.

For the construction of recombinant PR8 hemaglutinin expressing mutants,both full and headless form, a two step markerless red recombinationsystem as described by Tischer et al. (Tischer, B. K. et al, 2006,supra; Tischer, B. K. et al., Methods Mol. Biol. 2010, 634:421-30) (seealso FIG. 24) has been applied. Mutagenesis of full-length MCMV-BAC andΔm152-RAE1γ mCMV-BAC was performed in Escherichia coli strain DH10B,whereas excision of the selection marker was performed in Escherichiacoli strain GS1783.

Recombinant viruses were confirmed by restriction analysis (BamHI andHindIII restriction for Δm157-PR8-HA full-MCMV-BAC andΔm157-PR8-HA-full-Δm152-RAE-1γMCMV-BAC, and NsiI restriction enzyme forΔm157-PR8-HA-headless-MCMV-BAC and Δm157-Δm157-PR8-HAheadless-Δm152-RAE-1γMCMV-BAC)

In connection therewith it will be immediately acknowledged by a personskilled in the art that Δm157-PR8-HA full-MCMV is also referred toherein as MCMV-Δm157-HA and MCMV-HA.

Furthermore, Δm157-PR8-HA-full-Δm152-RAE-1γMCMV-BAC is also referred toherein as RAE-1γMCMV-HA or RAE-1γMCMV-Δm157-HA.

It will be understood that Δm157-PR8-HA-headless-MCMV is based onWT-MCMV, wherein influenza virus PR8 hemagglutinin (HA) headless formwas inserted into m157.

It will also be understood that Δm157-Δm157-PR8-HAheadless-Δm152-RAE-1γMCMV-BAC is based on RAE-1γMCMV, wherein influenzavirus PR8 hemagglutinin (HA) headless form was inserted into m157 and isalso referred to herein as Δm157-PR8-HA headless-Δm152-RAE-1γMCMV.

Generation of GAPINSATAM-Peptide Expressing Recombinant Viruses.

For the purpose of testing the vector capacity of Rae1γMCMV, aRAE-1γMCMV virus mutant has been constructed expressing Mycobacteriumtuberculosis H-2Db immunodominat epitope GAPINSATAM (SEQ.ID.NO: 16)“swapped” into the position of MCMV m164 immunodominant epitope ofWT-MCMV-BAC and RAE-1γMCMV-BAC by orthotopic peptide swap as describedin Lemmerman et al. (Lemmerman et al., 2010, supra). A schematicillustration of the cloning process is shown in FIG. 26.

(i) Design of Insert Containing Peptide Swap.

Primers were constructed in a way to replace the D^(d)-restrictedantigenic m164 peptide 167-AGPPRYSRI-175 (SEQ.ID.NO: 2) with theD^(b)-restricted Mtb32a (pepA) derived peptide 309-GAPINSATAM-318(SEQ.ID.NO: 16) (ref. UniProt O07175).

As primers m164 GAP fw:5′-cgcccgctgccacgatggcctggttgttgacggcccagaacatggcggtggccgagttgatcggggcgccgtcagcgccccaGCCAGTGTTACAACCAATTAACC-3′ (SEQ.ID.NO: 17), wherein lower case lettersrepresent homology region to m164 ORF in MCMV genome, underlined lettersin bold represent homology regions between primers, italic letters areI-SceI restriction site sequence and capital letters represent homologyto Tischer kanamycin cassette; and m164 GAP rv:5′-gccgttcggaaaggactactgtcggacgtggggcgctgacggcgccccgatcaactcggccaccgccatgTAGGGATAACAGGGTAATCGAT-3′ (SEQ.ID.NO: 18), wherein lower case letters representhomology region to m164 ORF in MCMV genome, underlined letters in boldrepresent homology regions between primers, italic letters are I-SceIrestriction site sequence and capital letters represent homology toTischer kanamycin cassette, were used. PCR was performed with thefollowing cycler conditions:

An initial step for 2 min at 98° C. for activation of HighFidelityPhusion DNA polymerase (New England BioLabs) was followed by 30 cyclesof 10 s at 98° C., 10 s at 60° C., and 60 s at 72° C. As DNA templateplasmid pEP-SaphAI provided by K. Tischer was used.

For the construction of recombinant mutants a two step markerless redrecombination system as described by Tischer et al. (Tischer, B. K. etal, 2010, supra) (see FIG. 24) has been applied. Mutagenesis offull-length MCMV-BAC and Δm152-RAE1γ mCMV-BAC was performed inEscherichia coli strain DH10B, whereas excision of the selection markerwas performed in Escherichia coli strain GS1783.

Construction of the ULBP2 Expressing HCMV Recombinant

The ULBP2 ORF was amplified using primers5′-GTCGGTACCGTCGCAGTCT-TCGGTCTGACCACCGTAGAACGCAGAGCTccaccATGGCAGCAGCCgccGCTACC-3′(SEQ.ID.NO: 19), wherein the underlined letters indicate homology to ntposition 182904-nt position 182857 (lower strand) of the mousecytomegalovirus sequence according to GenBank Accession No.: U68299.1;and 5′-cccGGATCCctctccTCAGATGCCAGGGAGGATGAAG-3′ (SEQ.ID.NO: 20), whereinthe Stop codon is indicated in bold and underlined and wherein small andlarge letters comprise additional details, e.g. Koszak sequence; and anULBP2 cDNA clone (Open Biosystems; Genbank accession number: BC034689),and was cloned via KpnI and BamHI restriction digest and ligation,between the MCMV major immediate-early promoter sequences and aKanamycin resistance (KanR) cassette flanked by FRT sites. The wholeinsert comprising promoter MCMV MIE (nt 183088 to 182903 (lower strand)of Genbank accession entry: U68299.1) and KanR was amplified withprimers 5′-GACACCGGGCTCCATGCTGACGTAGGTACCGACTGGGGTCAAAAGCCTttaaacggtactttcccatagc-3'(SEQ.ID.NO: 21), wherein the capitalized letters arehomologous to nucleotides 55134-55181 of the TB40E BAC (Genbank:EF999921.1) and5′-CTTATAGCAGCGTGAACGTTGCACGTGGCCTTTGCGGTTATCCGTTCAGgaacacttaacggctga-3′ (SEQ.ID.NO: 22), and inserted into the BAC-cloned genome ofthe HCMV strain TB40E (Genbank Accession Nr.: EF999921.1) (Sinzger C. etal., J Gen Virol. 2008 February; 89(Pt 2):359-68) by red-α, -β,-γ-mediated recombineering (Borst E. M. et al., 2007, supra) replacingthe UL16 ORF. The KanR cassette was excised by FLP recombinase. Correctinsertion was verified by restriction analysis and using BglII and NsiIand sequencing with primers 5′-GGCGATGCGGTATCGCGCACA-3′ (SEQ.ID.NO: 23)and 5′-GACACCTGTTCGTCCAGAATC-3′ (SEQ.ID.NO: 24).

Generation of a HCMV Vaccine Vector Expressing ULBP2 and the InfluenzaHemagglutinin Protein

The open reading frame (ORF) for influenza hemagglutinin (HA), accordingto Genbank accession number V01088, influenza A/PR/8 strain, is PCRamplified and cloned into plasmid vector (pUC19). Next to the HA ORF aPCR fragment is cloned carrying (i) a recognition site for themeganuclease I-SceI (ii) a sequence encoding kanamycin resistance, (iii)50 nt homologous to the end of the HA coding sequences and (vi) 20 nthomologous to sequences immediately downstream of the 3′-end of theUL110RF of the CMV strain TB40E (5′-TATATAGACTGAAGCGGAGT-3′ (SEQ.ID.NO:39); indicated as light grey colored rectangle in FIG. 31).

A PCR fragment is generated that includes the influenza HA ORF, the kanRcassette with the I-SceI site, the 50 nt homologous to the end of the HAORF, using the above described plasmid as template and primers thatprovide 50 nt of DNA sequences homologous to the sequences immediatelyupstream and downstream of the UL11 ORF in the TB40E genome,respectively. The sequence of the forward and reverse primers is:

UL11-HA-fw: (SEQ.ID.NO: 40)5′-CAGCTTTTGAGTCTAGACAGGGGAACAGCCTTCCCTTGTAAGACAGA ATGaagg-caaacctactggtcc-3′; and UL11-HA-rev: (SEQ.ID.NO: 41)5′-GAGTCGTTTCCGAGCGACTCGAGATGCACTCCGCTTCAGTCTATATA TCA-3′

Recombination between the BAC TB40E-ULBP2 carrying the TB40E genome withthe replacement of the UL16 ORF by a ULBP2 expression cassette (asdescribed above) and the PCR fragment is performed in E. coli strainGS1783 (Tischer, B. K. et al., Methods Mol Biol. 2010, 634:421-30)expressing the red α, -β, -γ genes as described in Borst et al., (BorstE M et al., Curr Protoc Immunol., 2007 May; Chapter 10: Unit 10.32.;Tischer, B. K. et al., 2010, supra). Bacterial clones carrying a BACwith a replacement of the US11 ORF with the HA-kanR cassette areselected on agar plates containing chloramphenicol (17 μg/ml) andkanamycin (30 μg/ml). Recombinant BACs are characterized by restrictionanalysis and sequencing as described in Borst et al. (Borst E M et al.,2007, supra). The kanR cassette is excised by en passant mutagenesis viacleavage of the BAC DNA with the I-SceI nuclease and red α, -β,-γ-mediated recombination in the E. coli strain GS1783 as described inTischer, B. K. et al., (Tischer, B. K. et al., 2010, supra).

More particularly, FIG. 31 schematically shows the construction ofrecombinant HCMV expressing ULBP2 and influenza HA generated asdescribed herein.

Infectious virus is generated by electroporation of human fibroblastswith the resulting BAC DNA as described in Borst et al. (Borst E M etal., 2007, supra).

Expression of the HA protein is tested in lysates of infected cells byimmunoblotting using an HA-specific antibody.

Reconstitution of BAC-Derived Recombinant Viruses.

The reconstitution of recombinant viruses by transfection of BAC plasmidDNA, as well as the routine elimination of BAC vector sequences, wasperformed in C57BL/6 primary mouse embryo fibroblasts (MEF) for MCMVmutants and in human foreskin fibroblasts (HFF) for HCMV mutants andverified by PCR.

For the reconstitution of MCMV mutants, recombinant constructs weretransfected using SuperFect reagent into 70% confluent MEFs prepared insix-well plate. Transfection solution was prepared according toSuperFect QIAGEN protocol. 7.5 μL of SuperFect was added to 150 μL totalvolume of recombinant construct mixed with DMEM. 10, 15 or 20 μl ofpurified BAC::DNA recombinant construct were used. Transfection solutionwas incubated for 10 min at room temperature to allow DNA complexesformation. During that period MEF were washed with 2 ml of PBS andoverlaid with 500 μL of 3% DMEM. After 10 minutes, 500 μL of 3% DMEM wasadded to transfection solution, mixed well and drop-by-drop applied tocell culture. After 2-3 h of incubation at 37° C., medium was removedand 4 mL of fresh 3% DMEM was added. 5-7 days post transfection plaquesappeared and supernatants were collected and used for second passage orstored at −70° C. till usage.

Verified BAC-vector-free virus clones were used to prepare high-titerstocks of sucrose gradient-purified viruses TB40_dUL16/ULBP2,MCMV-m164_List, also referred to herein as MCMVList and RAE1γMCMV-m164_List, also referred to herein as RAE-1γMCMVList, Δm157 HMIEPPR8 HA full mCMV, Δm157 HMIEP PR8 HA full Δm152 RAE1γmCMV, Δm157 HMIEPPR8 HA headless mCMV or Δm157 HMIEP PR8 HA headless Δm152 RAE1γ mCMV.

MCMV Production

MEFs were prepared in a Petri dish and grown to 70-80% confluence. 3 mLof supernatant containing virus particles was added to cell culture.When production was done from virus stock, 0.01 PFU per cell was addedin a volume of 6 mL 3% DMEM. After 3-4 h of incubation period at 37° C.,3% DMEM was added up to 30 mL. Cells were incubated at 37° C. for thetime of maximum infection (about 5 days) when all the cells rounded anddetached from the Petri dish, or could be removed by gently swirling themedium. After shaking off the cells, the mixture of cells and medium wastransferred into 50 mL tubes. Cells were separated from the medium bycentrifugation at 6400 g for 10 minutes. Supernatant was decanted intocentrifuge tubes. The virus was pelleted by centrifugation at 26000 gfor 90 minutes at 4° C. Supernatant was decanted, pellet resuspended inleftover medium and left at 4° C. overnight. Virus was laid over the 15%sterile sucrose/VSB cushion centrifuged 99 minutes at 4° C. and 52000 g.Supernatant was aspirated; pellet was overlaid with 300 mL of PBS andleft over night at 4° C. The pellet was resuspended, aliquoted andstored at −70° C.

Virus Growth Kinetics

MEFs were grown in 24-well plates and infected with 0.1 PFU/cell of thevirus. Virus was prepared in cold 3% DMEM. Media was removed from thecells and 200 mL of the virus suspension/well was added. Afterincubation period of 30 min at 37° C. 800 mL of warm 3% DMEM was addedin each well. After 1 h at 37° C. supernatant was taken for day 0, andchanged with 1 mL/well of warm 3% DMEM for all other time points.Supernatants were collected every day for seven days at the same time,centrifuged 4000 rpm/4 min to remove cells and cell debris and frozen at−20° C. till titration.

Preparation of Listeria monocytogenes Inoculum

The hemolytic EGD strain (serovar1/2a) of L. monocytogenes and therecombinant L. monocytogenes strain stably expressing chicken ovalbumin(aa134-387) (Zehn, D., et al. 2009, Nature 458(7235): 211-4.), alsoreferred to herein as OVA-Listeria, were grown in brain heart infusion(BHI) broth at 37° C. for 24 hours. The culture broth was centrifuged at3000×g for 5 minutes, and the pelleted bacteria were resuspended inphosphate buffered saline (PBS), pH 7.4. The optical density of thebacterial suspension was estimated using a spectrophotometer at 600 nm,and the numbers of CFU of L. monocytogenes were extrapolated from astandard growth curve. The actual number of CFU in the inoculum wasverified by plating on blood agar.

The actual number of CFU of OVA-Listeria in the inoculum was verified byplating on blood agar and BHI agar. Bacteria were grown in BHI broth toget enough material for inoculum. Blood agar was used for plating tocount the number in inoculum in addition to estimation withsprectrophotometer.

Animals, Infection and Lymphocyte Subsets Depletion.

BALB/c (H-2^(d)), C57BL/6 (H-2^(b)), interferon (IFN) type Ireceptor^(−/−) mice on 129 background (IFN-α/βR^(−/−)) and BALB/c(H-2^(d)) μMT/μMT^(−/−) mice (Hasan, M. et al., 2002, Eur J Immunol32(12):3463-71) were bred under specific-pathogen-free conditions at theCentral Animal Facility of the Faculty of Medicine, University ofRijeka. Animals handling, experimental procedures and administration ofanesthesia were performed in accordance with the guidelines contained inthe International Guiding Principles for Biomedical Research InvolvingAnimals. The Ethics Committee at the University of Rijeka approved allanimal experiments described within this report.

Unless otherwise indicated, mice were f.p. injected with 2×10⁵ PFU oftissue culture-derived MCMV at the age of 6 to 8 weeks. Neonatal micewere i.p injected with 500 PFU of MCMV 6 hours postpartum.

Infections were performed using WT-MCMV (Smith strain) and recombinantviruses constructed using WT-MCMV or RAE-1γMCMV backbone.

L. monocytogenes was injected when the log growth phase was achieved ina volume of 200 or 500 μL of pyrogen-free saline intravenously.

For challenge experiments, Listeria monocytogenes EGD serovar 1/2a wasused.

In vivo blocking of NKG2D, depletion of CD4⁺ T cells, CD8⁺ T cells andNK cells was performed by i.p. injection of monoclonal antibody (ratanti-mouse) to NKG2D (R&D Systems), CD4 (YTS 191.1), CD8 (YTS 169.4) andasialo-AGM1 (Wako Chemicals), respectively.

Determination of CFU in Mouse Organs

After dissection, organs (spleens and livers) were aseptically removedand homogenized in sterile PBS. After centrifugation for 5 min at 3000×gsupernatants were decanted, pellets were resuspended in 5 mL of colddistilled water and incubated on ice for 15 min to release intracellularbacteria. Bacterial counts were obtained by plating serial ten-folddilutions of each organ suspension on blood agar plates incubated at 37°C. for 24-48 h. Titres of L. monocytogenes were expressed as log₁₀of CFUper organ. In some experiments, small portions of the spleens and liverswere taken for histological analysis.

To determine organ OVA-Listeria burden, spleens and livers were removedfrom infected mice 4 days p.i. and homogenized separately in PBS,following incubation in distilled water. Serial ten-fold dilutions ofsuspensions were plated onto blood-agar and CFUs were determined after24-48 h incubation at 37° C. In some experiments, small portions of thespleens and livers were taken for histological analysis.

Determination of Virus Titers

To determine virus titer a standard plaque assay (Krmpotic, A. et al.,2005, J Exp Med 201(2):211-20) was performed. MEFs were prepared in48-well cell culture plates and grown to 70% confluence. Virus wastitrated in triplicates in log₁₀ dilutions series (starting from 10⁻³down to 10⁻¹⁰). After 1 h of incubation at 37° C., cells were layeredwith 500 μl of methylcellulose. Four days post infection virus plaqueswere counted and the PFU per mL was calculated according to formula:

Virus titer(PFU/ml)=number of plaques×V

-   -   V: dilution factor

To determine virus titer in mouse organs, the organs were dissected,transferred into 3% DMEM and frozen at −20° C. After ≧24 h organs werethawed slowly on ice and passed through the mesh. The mesh was rinsedwith 2 mL of 3% DMEM and organ homogenates were serially diluted infactor-10 steps for the virus plaque assay. MEFs were plated in 48 wellplates and grown close to confluence. The most of the cell culturemedium was removed in a way that the cell monolayer remained coveredwith fluid. A 100 μl of suspension of appropriate dilution was added induplicates. Plates were incubated for 30 min at 37° C. for virusadsorption and penetration and then centrifuged for another 30 min at760 g and 20° C. for enhanced penetration. After 30 min at 37° C. MEFswere covered with 0.2 ml of methylcellulose medium to prevent theformation of secondary plaques. Virus plaques were counted after 4 daysof cultivation.

Detection limit of the assay was extended to 1 PFU per organ homogenateas described previously (Polic, B. et al., 1998, supra).

Real-Time PCR.

Genomic DNA was extracted from 10 mg mouse tissue or 3001 blood usingWizard Genomic DNA Purification Kit (Promega), according to theinstruction manual, and dissolved in 100 μl of DNA Rehydration Solution.Viral genome was quantified by real-time PCR using the LightCyclersystem (Roche) and the LightCycler Fast Start DNA MasterPlus SYBR GreenI and analyzed by LightCycler data analysis software version 3.3.40.Primers ie4fwd (5′-TGACTTAAACTCCCCAGGCAA-3′; SEQ.ID.NO: 25) and ie4rev(5′-TAGGTGAGGCCATAGTGGCAG-3′; SEQ.ID.NO: 26), nucleotide positions:6692-6672 and 6592-6612, respectively (GenBank accession no. L06816),were chosen to amplify a segment of exon 4 of the ie1 gene. A cellulargene was detected with primers glra1fwd (5′-TGCCTGTTCTTTGCAGTCTGT-3′;SEQ.ID.NO: 27) and glra1rev (5′-AGTCGAGTGAAGGGTAACGAGC-3′; SEQ.ID.NO:28), nucleotide positions: 312-332 and 403-382, respectively (GenBankaccession no. X75832). Specificity of PCR products was determined bymelting curve analysis. Serial dilutions of pGEM-T Easy Vector (Promega)expressing partial MCMV ie1 gene and of DNA extracted from MEF were usedas standards to determine the MCMV genome copy numbers and the number ofcells, respectively. Tissues DNA samples from uninfected mice andmultiple samples without template served as negative controls. The PCRamplification efficiencies (E) for the ie1 and glra1 standard curves aswell as for both genes in a titration of sample DNA were calculatedaccording to the formula E=10^(−1/slope) (technical note no. LC11/2000;Roche) and differed by ΔE<0.05 in the reported experiments. Likewise,for each of the two genes tested, amplification efficacies differed byΔE<0.05 between a titration of sample DNA and the respective standardcurve. To determine sensitivity of QPCR detection of MCMV, genomic DNAsamples was spiked with serial dilutions of target plasmid pGEM-T EasyVector containing ie1 genomic sequence as template in the PCR aspreviously described (Wheat, R. L., et al. 2003, J Virol Methods112(1-2):107-13). Detection limit was found to be 6 copies of MCMV per10⁶ cells.

Isolation of Splenocytes

Spleen was cut with scissors into small pieces in 5 cm Petri dish andput on the cell strainer (70 or 100 mm) above 50 mL blue-cap. It waspressed with the pestle into blue-cap while washing with 10 mL 5% RPMI.Suspension was centrifuged 5 min at 1500 rpm (centrifuge C412, Jouan).Supernatant was decanted and 5 mL of 1× lysing buffer was added. Pelletwas resuspended and incubated for 5 min on ice. Lysis was stopped byaddition of 10 ml 5% RPMI. Suspension was centrifuged 5 min at 1500 rpm.Supernatant was poured out and the precipitate was resuspended in 10 mL10% RPMI. Suspension was strained through 70 or 100 mm cell strainer andsplenocytes were counted. Cell suspension was adjusted to the finalnumber of 10×10⁶ splenocytes/mL in 10% RPMI.

Interferon-γ Test

100 μl (1×10⁶ cells) of splenocytes suspension was put on 96 wellU-bottom and each sample was performed in duplicates. Peptides wereadded in concentration of 1 μg/sample and 100 μl of 10% RPMI was used asnegative control. Samples were incubated at 37° C. for 1 h. Brefeldin Awas added in 1:1000 ratio in volume of 10 μl per sample. Samples wereincubated at 37° C. for 4 h.

Adoptive Transfer of MCMV-Specific CD8⁺ T Cells.

Adoptive transfer experiment was performed as described previously(Holtappels, R. et al., 2008, J Infect Dis 197(4):622-9). In short,donors of CD8⁺ T cells were naïve or latently infected μMT/μMT^(−/−) (Bcell-deficient) mice, MCMV infected 6 months before the adoptivetransfer. Splenocytes from three donors per group were pooled and numberof MCMV-specific CD8⁺ T cells was assessed by combined staining with pp98, m164, m83, m84 and m04 MHC class I tetramers. Unfractionatedsplenocytes containing 10⁵ naïve CD8⁺ T cells or graded numbers ofMCMV-specific CD8⁺ T cells were i.v. transferred to recipient BALB/cmice immunocompromised with a single dose of 6 Gy γ-irradiation 12 hprior to adoptive transfer. Recipients were f.p. injected with 10⁵ PFUof WT-MCMV 6 h after the adoptive transfer. Viral titers in the spleenwere determined 12 days p.i. by plaque assay.

Flow Cytometry and Intracellular Staining.

After 4 h incubation, samples from IFNγ test were pooled to get 2×10⁶ ofcells per sample. Plates were centrifuged at 1500 rpm for 5 min. Cellswere washed with 200 μl of FACS medium and centrifuged at 1500 rpm for 5min. Supernatant was decanted, Fc block was added in volume of 25 μl andincubated 15 min on ice. αCD8 antibody was added in volume of 25 μl andincubated 20 min on ice. 150 μl FACS medium as added and centrifuged 5min at 1500 rpm. Supernatant was decanted and 150 μl of PBS was added.Plates were centrifuged 5 min at 4000 rpm. Supernatant was discarded and100 μl of 2% PFA in PBS was added. Plates were incubated 25 min at roomtemperature and washed two times in 100 μl PBS afterwards. Intracellularantibody was diluted in 0.1% saponin in FACS medium and incubated for 20min at room temperature. Cells were washed 2× in 150 μl of 0.05% saponinin PBS and resuspended in 200 μl of FACS medium.

Cells were put on 96 well and washed with 200 μl of FACS medium andcentrifuged at 1500 rpm for 5 min. Supernatant was decanted, Fc blockwas added in volume of 25 μl and incubated 15 min on ice. Primaryantibody was added in volume of 25 μl and incubated 20 min on ice. 150μl FACS medium as added and centrifuged 5 min at 1500 rpm. Supernatantwas decanted and secondary antibody was added in volume of 501 andincubated for 20 min on ice. Cells were washed in FACS medium andcentrifuged at 1500 rpm for 5 min. Supernatant was discarded and cellswere resuspended in 200 μl of FACS medium.

The H2^(b)-restricted SIINFEKL pentamer was purchased from Proimmune.SIINFEKL peptides were synthesized to a purity of >95% by Jerini PeptideTechnologies.Custom MCMV-specific H-2^(d) and H-2^(b) class I restrictedantigenic peptide synthesis to a purity of >80% was performed by JeriniPeptide Technologies. Tetramers were synthesized by the NIH tetramercore facility (http://www.niaid.nih.gov/reposit/tetramer/overview.html).Various fluorescently conjugated antibodies were used (CD8α (53-6.7),CD27 (LG.7F9), CD62L (MEL-14), CD122 (5H4), CD127 (A7R34), KLRG-1 (2F1),NKG2A/C/E (20D5), NKG2D (M1-6), CTLA-4 (UC10-4B9), PD-1 (J43), IL-2(JES6-SH4), IFN-γ (XMG1.2), TNF-α (MP6-XT22), CD3β (H57-597), CD11c(N418), NKp46 (29A1.4), PDCA-1 (eBio927), RAE-1γ (CX1), RAE-1αβδε(199205), MULT-1 (237104), H60 (205326). An in vitro assay to detectcytokine production and degranulation was performed as previouslydescribed (Cicin-Sain, L. et al., 2007, supra). In short, splenocyteswere resuspended in complete RPMI 1640 supplemented with 10% FCS andstimulated with 1 μg of peptides IE1/m123 (¹⁶⁸YPHFMPTNL¹⁷⁶; SEQ.ID.NO:1), also known as a pp 89 derived, m164 (₁₆₇AGPPRYSR₁₁₇₅; SEQ.ID.NO: 2),IE3 (⁴¹⁶RALEYKNL⁴²³; SEQ.ID.NO: 29), m139 (⁴¹⁹TVYGFCLL⁴²⁶; SEQ.ID.NO:30), M45 (⁹⁸⁵HGIRNASFI⁹⁹³; SEQ.ID.NO: 31) or M38 (³¹⁶SSPPMFRV³²³;SEQ.ID.NO: 32) for 6 h at 37° C. with brefeldin A (eBioscience) addedfor the last 4 hours of stimulation. For degranulation assay, CD107amonoclonal antibody (D4B) and monensin (eBioscience) were added to thecultured cells during peptide stimulation. For DC population analysis,splenocytes were digested by collagenase D (Roche) as describedpreviously (Robbins, S. H. et al., 2007, supra). All samples wereacquired by FACSAria (BD) and analyzed with FlowJo software (Tree Star).

In Vivo Cytotoxicity Assay.

Splenocytes were isolated from the spleens of uninfected BALB/c mice andloaded with 1 μg/mL of listeriolysin peptide ⁹¹GYKDGNEYI⁹⁹ per 6×10⁷cells for 1 h at 37° C. before labeling with CFSE. LLO peptide-pulsedsplenocyte targets were labeled with a low concentration (0.5 μM) ofCFSE (Invitrogen), whereas control targets, not pulsed with a peptide,were labeled with a high concentration (5 μM) of CFSE for 10 minutes at37° C., followed by being washed and incubated for an additional 30 minat 37° C. Differentially CFSE-labeled cells were washed, mixed in equalratios and a total of 1×10⁷ cells per mouse was transferredintravenously into MCMVList or RAE1γMCMVList-infected or uninfectedmice. Spleens were harvested 6 h later and the survival of thetransferred splenocytes was analyzed by flow cytometry.Percentage-specific lysis of CFSE-labeled target cells was calculated asfollows:

(1−[r uninfected control mouse/r infected test mouse])×100 wherer=(frequency of unpulsed targets/frequency of peptide-pulsed targets).

Sequence Analysis of RAE-1γ.

Organ homogenates from μMT/μMT B cell-deficient mice with recurrentinfection were serially diluted 2-fold across 96-well trays and added toMEF cultures in 96-well trays. Wells showing viral cytopathic effectderived from a single plaque were harvested for preparation of virusstocks. The RAE-1γ ORF was PCR amplified by using purified viral DNA.MEFs were infected with the recovered viruses and whole genomic DNA wasextracted using a DNeasy blood and tissue kit (Qiagen). The region ofinterest was amplified by PCR with primers m152fw GTGTATGTGGCCCGACGGGCGG(SEQ.ID.NO: 33) and m152rv CGCGGGCTACTCCCGAAAGAGTAACATC (SEQ.ID.NO: 34).The amplificate was sequenced (3130 genetic analyzer, AppliedBioscience) using the primers m152fw GTGTATGTGGCCCGACGGGCGG (SEQ.ID.NO:33, m152rv CGCGGGCTACTCCCGAAAGAGTAACATC (SEQ.ID.NO: 34), RAEfwATGGCCAAGGCAGCAGTGAC (SEQ.ID.NO: 35) and RAErvTGCTCGACCTGAGGTAATTATAACCC (SEQ.ID.NO: 36). Sequences were aligned tothe RAE-1γ ORF of the input virus using Vector NTI 11 (Invitrogen).

Quantification of MCMV-Specific Antibody and Serum IFN-α Level by ELISA.

Serum MCMV-specific IgG titers were determined by ELISA as previouslydescribed (Jonjic, S. et al., 1990, J Virol 64(11):5457-64). Serumlevels of IFN-α were determined by ELISA KIT for IFN-α (PBL BiomedicalLaboratories) according to the manufacturer's instructions.

Histological Methods

Hematoxylin-Eosin (HE) staining on paraffin sections was performed asdescribed in the following: Paraffin sections were deparaffined bywashing slides 2×5 min in xylene, 2×3 min in 100% ethanol, 2×3 min in95% ethanol and 2×5 min in PBS. Slides were stained with hematoxylin 1min and washed for 5 min in distilled water. Eosin staining wasperformed for 30 sec and slides were washed as follows: 5 min 70%ethanol, 5 min 80% ethanol, 5 min 90% ethanol, 5 min 100% ethanol, 2×5min xylene. Slides were mounted using Aquatex.

Anti-CD3 staining on paraffin sections was performed as described in thefollowing: Paraffin sections were deparaffined by washing slides 2×5 minin xylene, 2×3 min in 100% ethanol, 2×3 min in 95% ethanol and 2×5 minin PBS. Endogenous peroxidase was blocked for 30 min in hydrogenperoxide solution. Slides were washed 2×5 min in PBS. Antigen retrivalwas performed in citrate buffer at 800 w for 4 min and at 400 W at 15min. Slides were cooled at room temperature for 20 min and then washed2×3 min in distilled water and 2×5 min in PBS. Unspecific staining wasblocked by incubation in serum for 20 min. Biotin was blocked by 15 minincubation in avidin block solution following 15 min biotin blocksolution. Primary antibody was diluted in 1% BSA-TBS and incubated for 1h. Slides were washed 2×5 min in PBS. Secondary antibody biotin labeledwas diluted in 1% BSA-TBS and incubated for 30 min. Slides were washed2×5 min in PBS, incubated with streptavidin-peroxidase for min andwashed 3×5 min in PBS. AEC was incubated for 10 min, slides were washed3×1 min in distilled water, 10 sec in hematoxylin, 10 min under tapwater, 1 min in distilled water and mounted using Aquatex.

Histopathology and Immunohistochemistry.

Sections from formalin-fixed, paraffin-embedded spleens were stainedwith hematoxylin and eosin (both Thermo Scientific). CD3-expressinglymphocytes on paraffin sections were visualized by anti-CD3 (SP7)(Abcam), followed by biotinylated goat anti-mouse immunoglobulin G (IgG)antibody (BD Pharmingen, San Diego, Calif.) and avidin-biotin-peroxidasecomplex (Roche Applied Science, Manheim, Germany) staining.Counterstaining was performed with hematoxylin. Slides were analyzed onan Olympus BX40 microscope, and images were acquired by Olympus digitalcamera (C-3030).

Statistics.

Statistical significance was calculated by unpaired two-tailed Student'st test using Prisma 4 software and/or GraphPad Prism 5 software(GraphPad Software). The significant differences between tested groupsare indicated with star symbols as follows: p<0.05 (*), p<0.01 (**) andp<0.001 (***). Statistical analyses of the virus titers were done byMann-Whitney U test. Flow cytometry analysis was performed using BDFACSDiva software.

Example 2 Generation and In Vitro Characterization of a Recombinant MCMVExpressing the NKG2D Ligand RAE-1γ

To study how the expression of NKG2D ligand by MCMV influencesimmunobiology of this virus infection, the present inventor designed arecombinant virus, referred to herein as RAE-1γMCMV that expressesRAE-1γ. RAE-1γMCMV was constructed by replacing the m152 ORF in the(BAC-cloned) MCMV genome with a cassette comprising the RAE-17 ORF undercontrol of the HCMV immediate-early promoter (FIG. 1A).

More particulary, FIG. 1A shows the HindIII cleavage map of the MCMVgenome at the top with the genomic region encoding the m152 ORF below.In order to construct RAE-1γMCMV the m152 ORF was replaced by anexpression cassette (bottom) comprising the HCMV major immediate earlypromoter (CMV-P), the RAE-1γ ORF (RAE-1γ) and the SV40 polyadenylationsignal sequence (pA).

RAE-1γMCMV replication was assessed in a multistep growth kinetics assayand was compared to WT-MCMV replication. The results are shown in FIG.8A.

More particularly, FIG. 8A shows virus titers determined in supernatantsof MEF which were infected with RAE-1γMCMV (triangle) or WT-MCMV(squares) at 0.1 PFU per cell. Supernatants were harvested at timepoints p.i. as indicated on the x-axis and virus titers were determinedby plaque assay. Means±standard errors for the representative of 3experiments are shown. The virus titer is depicted on the y-axis as log10 PFU per milliliter, ml.

It may be taken therefrom that RAE-1γMCMV replication was comparable toreplication of WT-MCMV

Infection of SVEC4-10 cells, an endothelial cell line that does notexpress RAE-1γ, with the recombinant MCMV resulted in cell surfaceRAE-1γ expression, which may be taken from FIG. 1B.

More particularly, FIG. 1B shows FACS analysis of SVEC4-10 cells (upperpanel), NIH 3T3 cells (lower panel) and MEF (middle panel) which wereinfected with indicated viruses, i.e. WT-MCMV, Δm152-MCMV or RAE-1γMCMV,or left untreated and which were analyzed 12 h later for the surfaceexpression of RAE-1γ by staining with the anti-RAE-1γ antibody, followedby PE-conjugated goat anti-rat IgG. Cells incubated with the secondaryantibody in the absence of the primary antibody were used as negativecontrol (thin line). Each histogram represents 10,000 gated propidiumiodide-negative cells. On the x-axis the relative intensity of PE(Phycoerythrin) signal is shown. The y-axis depicts the cell number aspercentage of total cells. One of two similar experiments is shown.

It may also be taken from FIG. 1B that WT-MCMV infection down-regulatesendogenous RAE-1γ which was prevented by the deletion of m152 from theMCMV genome (Δm152 MCMV). Introduction of RAE-1γ to the Δm152-MCMVresulted in RAE-1γ over-expression on the surface of infected cells.

FIG. 8B shows the surface expression of NKG2D ligands on NIH 3T3 (leftpanel), SVEC 4-10 (middle panel) and B12 cells (right panel) which wereinfected with indicated viruses at 3 PFU per cell or left uninfected,and 12 h later analyzed for the surface expression of NKG2D ligands bystaining with the biotinylated anti-H60 antibody, anti-MULT-1 antibodyor anti-RAE-1αβ antibody, followed by PE-conjugated goat anti-rat IgG orPE-labeled streptavidin. Cells incubated with the secondary antibody inthe absence of the primary antibody were used as negative control (thinline). Each histogram represents 10,000 gated propidium iodide-negativecells. On the x-axis the relative intensity of secondary antibody signalis shown. The y-axis depicts the cell number as percentage of totalcells.

As may be taken from FIG. 8B RAE-1γMCMV infection did not change thepattern of cell surface expression of other NKG2D ligands compared toΔm152-MCMV.

Altogether, these data indicate that RAE-1γ insertion into the MCMVgenome had no effect on virus replication in vitro and resulted in theexpression of RAE-1γ on the surface of infected cells. Furthermore itmay be taken from the above that RAE-1γMCMV is attenuated in vivo in anNKG2D-dependent manner.

Example 3 RAE-1γMCMV is Strongly Attenuated In Vivo and Fails toEstablish Persistent Infection in Salivary Gland

Adult BALB/c mice were injected with RAE-1γMCMV, WT-MCMV or Δm152-MCMVto study whether expression of the NKG2D ligand by the MCMV influencesvirus control in vivo. In agreement with previous results (Krmpotic, A.et al., 2002, Nat Immunol 3:529-535), at day 3 post infectionreplication of Δm152-MCMV was attenuated in an NKG2D-dependent manner ascompared to WT-MCMV. Introduction of RAE-1γ to the Δm152-MCMV genomefurther attenuated viral replication and resulted in significantly lowerviral titers in all tested organs as compared to Δm152-MCMV and WT-MCMVas may be taken from FIG. 1C.

More particularly, FIG. 1C shows the virus titer in lungs (upper panel)or spleen (lower panel) of untreated BALB/c mice or BALB/c mice treatedwith blocking anti-NKG2D monoclonal antibody which were i.v. injectedwith 10⁵ PFU of the indicated viruses, i.e. WT-MCMV (white circles),Δm152-MCMV (grey circles) or RAE-1γMCMV MCMV (black circles). Viraltiters were determined by plaque assay in lungs and spleen 3 days postinfection, abbreviated herein preferably as d p.i. or days p.i. Virustiter in organ of individual mice (circles) and median values(horizontal bars) are shown. One of two similar experiments is shown.The virus titer is shown on the y-axis as log₁₀ PFU.

The observed attenuation was NKG2D-dependent and was abolished byadministration of anti-NKG2D blocking antibodies that restoredRAE-1γMCMV titers almost to the WT-MCMV level.

FIG. 1D shows a diagram indicating virus load in salivary glands atdifferent time points as indicated on the x-Axis of BALB/c mice whichwere f.p. injected with 2×10⁵ PFU of WT-MCMV (white), Δm152-MCMV (grey)or RAE-1γMCMV (black). Viral titers were determined by plaque assay atthe indicated time points post infection. Virus titer in salivary glandsof individual mice (circles) and median values (horizontal bars) areshown. Virus titer is shown on the y-axis as log₁₀ PFU per salivarygland. DL means detection limit and is indicated by the dashed line. Oneof two similar experiments is shown.

It may be taken therefrom that the salivary glands remain persistentlyinfected with MCMV long after productive virus replication is terminatedin other tissues (Reddehase, M. J. et al., 1994, J Exp Med179(1):185-93; Jonjic, S. et al., 1989, J Exp Med 169(4):1199-212). NKcells and CD4⁺ T cells are essential for virus clearance in the salivaryglands and prevention of horizontal virus spread (Jonjic, S. et al.,1989, supra; Campbell, A. E. et al., 2008, Med Microbiol Immunol197(2):205-13). The present inventor therefore compared the virus titersin salivary glands 15, 60 and 150 days after RAE-1γMCMV, WT-MCMV andΔm152-MCMV infection. In contrast to a high-titer persistent virusreplication in WT-MCMV infected mice, no infectious virus was detectedin salivary glands following RAE-1γMCMV infection (FIG. 1D).

Although Δm152-MCMV reached slightly lower virus titers compared toWT-MCMV, replication kinetics of these two viruses in salivary glandswere similar. The present inventor next determined whether markeddifferences between RAE-1γMCMV and WT-MCMV replication are reflected inthe kinetics of viral clearance from blood and viral genome load intissue during latency.

FIG. 1E shows the copies of viral genomes in blood (upper panel), liver(middle panel) and salivary gland (lower panel) of BALB/c mice whichwere f.p. injected with 2×10⁵ PFU of WT-MCMV (white), Δm152-MCMV (grey)or RAE-1γMCMV (black). Viral genome load was determined by qPCR atdifferent time points post infection as indicated on the x-axis. Viralgenome load in organs of individual mice (circles) and median values(horizontal bars) are shown as log₁₀ viral genome copies per 10⁵ cells.DL means detection limit and is indicated by the dashed line. One of twosimilar experiments is shown.

It may be taken therefrom that unlike WT-MCMV infection in which viralDNA was maintained in the blood for prolonged period of time (Balthesen,M. et al., 1993, J Virol 67(9):5360-6), viral DNA was cleared from theblood of RAE-1γ MCMV infected mice by day 45. At that time, RAE-1γMCMVDNA remained in organs, but the viral load was reduced to a barelydetectable level or in some cases, to below the limit of detection.Viral DNA load in Δm152-MCMV infected mice corresponded to infectiousvirus titers (FIG. 1E).

To study how the expression of NKG2D ligand by the MCMV affects viruscontrol in mice with constitutively more efficient NK cell response, thepresent inventor injected C57BL/6 with RAE-1γMCMV, WT-MCMV or Δm152MCMV. MCMV resistance of C57BL/6 mice is due to the expression of Ly49Hactivating receptor on NK cells, which recognizes virally encodedprotein m157 (Arase, H. et al., 2002, Science 296(5571):1323-6; Smith,H. R. et al., 2002, Proc Natl Acad Sci USA 99(13):8826-31).

FIG. 9A shows the virus load in lungs (upper panel) and spleen (lowerpanel) of untreated C57BL/6 mice or C57BL/6 mice injected with blockinganti-NKG2D antibody, as indicated on the x-axis, which were i.v.injected with 5×10⁵ PFU of WT-MCMV (white circles), RAE-1γMCMV (blackcircles) or Δm152-MCMV (grey circles). Viral titers were determined inspleen 3 d p.i. by plaque assay and are indicated on the y-axis as log₁₀PFU/lungs or spleen, respectively.

It may be taken therefrom that, similarly to results in MCMV-sensitiveBALB/c mice, RAE-1γMCMV reached significantly lower titers compared toWT-MCMV and Δm152 MCMV, hence RAE-1γMCMV is attenuated in Ly49H⁺ C57BL/6mice.

Thus, NKG2D-mediated control of RAE-1γMCMV was not covered by NK cellactivation via Ly49H as in the case following infection with MCMV mutantlacking m152 only, which may be taken from FIG. 9 A. Taken together,expression of RAE-1γ by MCMV resulted in a dramatic attenuation of virusreplication in different organs and a lower latent viral DNA load.

Example 4 RAE-1γMCMV is Attenuated Even in Neonatal Mice

Neonatal mice are highly sensitive to MCMV infection and intraperitoneal(i.p) injection even with low dose of cell culture-derived virus resultsin significant morbidity and mortality. Mice that survive MCMV infectionestablish a disseminated, high-titer virus replication and long-lastingpersistent infection in salivary glands (Reddehase, M. J. et al., 1994,supra). To test RAE-1γMCMV replication in neonatal mice, newborn animalswere injected i.p. with 500 PFU of RAE-1γMCMV or WT-MCMV.

The results may be taken from FIG. 2A.

More particularly, FIG. 2A is a diagram showing virus load in spleen(upper left panel), liver (upper right panel), lungs (lower left panel)and salivary gland (lower right panel) of neonatal BALB/c mice whichwere i.p. injected with 500 PFU of RAE-1γMCMV (black circles) or WT-MCMV(white circles) 6 hours post partum. Viral titers were determined byplaque assay at the time points post infection indicated at the x-axis.Viral titer of organs of individual mice (circles) and median values(horizontal bars) are shown as log₁₀ PFU per gram (g) tissue. DL meansdetection limit and is indicated by the dashed line. One representativeof two experiments is shown.

It may be taken therefrom that during the first 5 days of infection bothviruses replicated to comparable titers, but starting from day 7RAE-1γMCMV replication was significantly reduced in all tested organs(FIG. 2A).

Productive RAE-1γMCMV infection was cleared by day 11 in spleen andliver and by day 19 in lungs and even in salivary glands. By contrast,around that time WT-MCMV replication in salivary glands and lungs wereplateau levels (FIG. 2A) and productive infection continued for severalmonths (Reddehase, M. J. et al., 1994, supra) and data not shown).

Accordingly, RAE-1γMCMV is attenuated in neonatal mice.

Similarly to results obtained in adult mice, Δm152-MCMV replication wasattenuated compared to WT-MCMV but not to the level of RAE-1γMCMVattenuation. Furthermore ca. 3 weeks after infection, Δm152-MCMV stillreplicated to high titers in salivary glands, which may be taken fromFIG. 10.

FIG. 10 shows the viral load in spleen, liver, lungs and salivary gland,as indicated from left to right, of neonatal BALB/c mice which were i.p.injected with 500 PFU of RAE-1γMCMV (black circles), WT-MCMV (whitecircles) or Δm152-MCMV (grey circles) 6 h post partum. Viral titers weredetermined by plaque assay at day 7 p.i. (upper panel) or day 19 p.i.(lower panel). The virus titer of individual mice (circles) and medianvalues (horizontal bars) are shown as log₁₀ PFU per gram tissue on they-Axis. DL means detection limit and is indicated by the dashed line.

It may be taken therefrom that RAE-1γMCMV is attenuated compared to MCMVlacking the m152 gene only, namely Δm152-MCMV, in new-born mice.

Attenuated RAE-1γMCMV replication in neonates led to a lower load ofviral DNA in various organs, while prolonged, high-level WT-MCMVreplication resulted in higher load of viral DNA in organs, which may betaken from FIG. 2B.

More particularly, FIG. 2B shows copies of viral genome in spleen (upperleft panel), liver (upper right panel), lungs (lower left panel) andsalivary gland (lower right panel) of neonatal BALB/c mice which werei.p. injected with 500 PFU of RAE-1γMCMV (black circles) or WT-MCMV(white circles) 6 hours post partum, determined by qPCR at the timepoints post infection, p.i., indicated on the x-axis. Virus genomecopies of organs of individual mice (circles) and median values(horizontal bars) are shown on the y-axis as log₁₀ virus genome copiesper 10⁶ cells. DL means detection limit and is indicated by the dashedline. One representative of two experiments is shown.

As may be taken from the above, RAE-1γMCMV infection in neonates ischaracterized by attenuated virus replication, shorter duration of theproductive infection and subsequent lower virus DNA load as compared tothe WT-MCMV.

Example 5 Efficient Priming and Maintenance of Adaptive Immune Responseafter RAE-1γMCMV Infection

To test whether the RAE-1γMCMV attenuation impacts on the adaptiveantiviral immune response, adult BALB/c mice were footpad injected with2×10⁵ PFU of RAE-1γMCMV, WT-MCMV or Δm152 MCMV. The kinetics of thevirus specific T cell response was followed by use of MHC class Itetramers loaded with MCMV peptides (Holtappels, R. et al., 2008, MedMicrobiol Immunol 197(2):125-34).

The results are shown in FIG. 3A.

More particularly, FIG. 3A shows the percentage of m164-(left panel) andIE1/m123-(rightpanel) tetramer-specific CD8+ T cells of BALB/c micewhich were f.p. injected with 2×10⁵ PFU RAE-1γMCMV (black circles),WT-MCMV (white circles) or Δm152-MCMV (grey circles). Splenocytes wereisolated at different time p.i. as depicted on the x-axis. Thesplenocytes were stained with IE1/m123 or m164 MHC class I tetramers andanti-CD8 antibody. The percentage of tetramer-specific CD8⁺ T cells forindividual mice (circles) and median values (horizontal bars) are shownon the y-axis.

The CD8⁺ T cell response was dominated by IE1/m123-specific andm164-specific cells, while the response to the 4 other studied epitopes(m04, M83, M84, M45) was low or below the level of detection (FIG. 3Aand data not shown).

Following infection with either of three viruses the m164-specific CD8⁺T cells displayed comparable stable memory kinetics. By contrast,immunoinflation of IE1/m123-specific T cells in spleen at 9 months p.i.was less prominent following RAE-1γMCMV and Δm152-MCMV than afterWT-MCMV infection (Holtappels, R. et al., 2000, J Virol74(24):11495-503). The kinetics of the antiviral CD8⁺ T cell response inthe blood closely reflected that in spleen (data not shown). Thephenotypic and functional properties of virus-specific CD8⁺ T cells weresimilar following RAE-1γMCMV, WT-MCMV and Δm152-MCMV infection, whichmay be taken from FIG. 3B.

More particularly, FIG. 3B shows the percentage of IE1/m123-specificCD8⁺ T cells of splenocytes isolated 9 mo p.i. from BALB/c mice whichwere f.p. injected with 2×10⁵ PFU RAE-1γMCMV (black bars), WT-MCMV(white bars) or Δm152-MCMV (grey bars). Splenocytes were stained withIE1/m123 MHC class I tetramer, anti-CD8 antibody and antibodies to cellsurface molecules indicated on the x-axis. The percentage ofIE1/m123-specific CD8⁺ T cells displaying Tem (T_(EM) or effectormemory) or Tcm (T_(CM) or central memory) phenotype (left panel) and thepercentage of Tem and Tcm expressing indicated cell surface moleculesare shown (right panels). Error bars show the means±standard errors ofthe means.

Between 60 and 75% of IE1/m123-specific and m164-specific CD8⁺ T cellsin spleen and blood retained effector memory phenotype (TEM) up to 9months after the infection. It is important to note that the expressionof NKG2D, a CD8⁺ T cell costimulatory receptor, was essentiallyidentical following both RAE-1γMCMV and WT-MCMV infection, which may betaken from FIG. 3C.

More particularly, FIG. 3C shows a representative histogram of the FACSanalysis of surface expression of NKG2D on IE1/m123-specific CD8⁺ Tcells in spleen 9 month after f.p. injection of 2×10⁵ PFU WT-MCMV(filled histogram) or RAE-1γMCMV (dotted line). Tetramer negative CD8⁺ Tcells are indicated as a dashed line.

Also, the inhibitory receptors PD-1 and CTLA-4, described to beassociated with T cell exhaustion during persistent infections (Wherry,E. J., and Ahmed, R., 2004, J Virol 78(11):5535-45) were not upregulatedon memory CD8⁺ T cells and the T cells remained fully functionalthroughout latent RAE-1γMCMV and WT-MCMV infection, which may be takenfrom FIG. 3D and Figure E.

More, particularly, FIG. 3D shows a series of dot plots of FACS analysisof splenocytes from BALB/c mice which were f.p. injected with 2×10⁵ PFUWT-MCMV (upper row) or RAE-1γMCMV (lower row), wherein the splenocyteswere isolated 9 month post infection (mo p.i) and stained with theindicated tetramers, i.e. IE1/m123- or m164-tetramer, (left panel) orstimulated with the indicated peptides, i.e. IE1/m123- or m164-peptide,and stained for IFN-γ production (right panel). The representative dotplots are gated on CD8⁺ T cells of 3 mice per group are shown. Numbersindicate means. One of two similar experiments is shown.

FIG. 3E shows a series of dot plots showing FACS analysis of splenocytesof mice which were f.p. injected with 2×10⁵ PFU WT-MCMV (upper row) orRAE-1γMCMV (lower row). The splenocytes were isolated 9 mo p.i. andstimulated with the indicated peptides, i.e. IE1/m123- or m164-peptide,in the presence of the αCD107a antibody and co-stained for IFN-γ andTNF-α production. The y-axis indicates the intensity of the staining forIFN-γ production. The x-axis indicates the intensity of the staining forTNF-α production on the left panel and the intensity of the staining forCD107a on the right panel. The representative dot plots are gated onCD8⁺ T cells of 3 mice per group are shown. Numbers indicate means. Oneof two similar experiments is shown.

It may be taken therefrom that at each time point analyzed, thepercentage of CD8⁺ T cells detected by tetramer staining was similar tothe percentage of CD8⁺ T cells secreting IFN-γ upon stimulation with aviral antigenic peptide in vitro (see FIG. 3D) and most of the cellssimultaneously secreted TNF-α, but not IL-2 (see FIG. 3E and data notshown) and extruded cytotoxic granules, i.e. externalized CD107a (seeFIG. 3E).

It may be also taken from the above that kinetics and phenotype ofMCMV-specific memory CD8⁺ T cells in RAE-1γMCMV, WT-MCMV and Δm152-MCMVinfected mice are comparable.

Interestingly, in C57BL/6 mice frequency of MCMV-specific CD8⁺ T cellsat early time point after RAE-1γMCMV infection was even higher comparedto WT-MCMV, which may be taken from FIG. 9 B.

More particularly, FIG. 9B shows the percentage of IFN⁺CD8⁺ T cells ofsplenocytes from C57BL/6 mice which were f.p. injected with 2×10⁵ PFU ofRAE-1γMCMV (black circles) or WT-MCMV (white circles). Splenocytes wereisolated at different time points p.i, as indicated on the x-axis, andstimulated with the indicated peptides, namely m139-peptide according toSEQ.ID.NO: 30 (upper left panel), M45-peptide according to SEQ.ID.NO: 31(upper right panel), M38 peptide according to SEQ.ID.NO: 32 (lower leftpanel) or IE3-peptide according to SEQ.ID.NO: 29 (lower right panel),and stained for IFN-γ production. The percentage of IFN⁺CD8⁺ T cells forindividual mice (circles) and median values (horizontal bars) are shownas percentage of IFN⁺CD8⁺ T cells on the y-axis.

Similar priming capacity and the frequency of virus specific CD8⁺ Tcells after infection with RAE-1γMCMV or WT-MCMV in spite of dramaticdifferences in the load of infectious virus in their tissues, promptedthe present inventor to test whether this can be explained bydifferential effect of RAE-1γMCMV and WT-MCMV on dendritic cells (DC) invivo. MCMV infection results in a reduction of conventional DCs (cDC) inBALB/c mice which can be prevented by efficient antiviral NK cellresponse in C57BL/6 strain (Robbins, S. H. et al., 2007, supra; Andrews,D. M. et al., 2010, J Exp Med 207(6):1333-43).

To test how the vaccine virus affects DCs in vivo the present inventorcompared DC subsets following RAE-1γMCMV and WT-MCMV injection in BALB/cmice.

The results are shown in FIG. 11.

More particularly, FIG. 11 shows representative dot plots of FACSanalysis of splenocytes which were isolated from naïve BALB/c mice(uninfected) or BALB/c mice i.v. injected with 2×10⁵ PFU of RAE-1γMCMVor WT-MCMV, as indicated, 3 days p.i. and analyzed for the frequency ofCD11b cDCs (CD11c^(hi)CD8α⁻) and CD8αcDCs (CD11c^(hi)CD8α⁺) within theNKp46⁻ TCRβ⁻ population. Numbers in dot plots represent the percentageof CD11b cDCs and CD8α cDCs within the total splenocyte population for arepresentative animal from a group of three mice.

It may be taken therefrom that while a marked reduction of cDCs occurredat early time after WT-MCMV infection, both CD11b and CD8α subsets ofcDC were preserved following RAE-1γMCMV infection (see FIG. 11).

As reported by others (Robbins, S. H. et al., 2007, supra) the frequencyof cDC in spleen of infected mice inversely correlated with type Iinterferons levels in sera of infected mice. At day 2 post infection theaverage level of IFN-α in sera was significantly higher after WT-MCMV(5212±1266 pg/ml) as compared to RAE-1γMCMV infection (1459±840 pg/ml).Thus, an efficient early control of RAE-1γMCMV resulted in preservationof cDCs, possibly by preventing an overwhelming production of type IIFNs, providing optimal conditions for priming of MCMV-specific T cells.

In vivo antiviral effector activity of MCMV-specific memory CD8⁺ T cellsgenerated following RAE-1γMCMV and WT-MCMV infection was compared byprophylactic adoptive transfer into immunodepleted MCMV-infectedrecipient mice. The result is shown in FIG. 4A and FIG. 4B.

More particularly, FIG. 4A shows virus titers of BALB/c mice infectedafter transfer of memory CD8⁺ T from latently infected μMT/μMT Bcell-deficient mice.

Donors of memory CD8⁺ T cells were taken from μMT/μMT B cell-deficientmice either naïve (grey circles) or latently infected with RAE-1γMCMV(black circles) or WT-MCMV (white circles) at least 6 mo p.i.Splenocytes from three donors per group were pooled and the number ofMCMV specific CD8⁺ T cells was assessed by combined staining with pp98,m164, m83, m84 and m04 MHC class I tetramers. 10⁴ naïve CD8⁺ T cells orgraded numbers of MCMV-specific CD8⁺ T cells, as indicated on thex-axis, were i.v. transferred to recipient BALB/c mice immunocompromisedby 6 Gy γ-irradiation. Recipients were f.p. injected with 10⁵ PFU ofWT-MCMV 6 h after the cell transfer.

Viral titers in spleen were determined 12 d p.i. by plaque assay and areshown on the y-axis as log₁₀ PFU per spleen. Titers of individual mice(circles) and median values (horizontal bars) are shown. Ø means notransfer. DL means detection limit and is indicated by the dashed line.

FIG. 4B shows the percentage of IE1/m123 MHC class I tetramer-positivecells per CD8+ T cells of mice infected as described in FIG. 3 whichwere i.p. challenged with 10⁵ PFU of salivary gland derived MCMV, alsoreferred to herein as SGV, 6 mo p.i. Lymphocytes were isolated fromblood (middle panel), spleen (left panel) and liver (right panel) atdifferent time points after the challenge as indicated on the x-axis andstained with IE1/m123 MHC class I tetramer and anti-CD8 antibody. Thepercentage of IE1/m123-specific CD8⁺ T cells for individual mice(circles) and median values (horizontal bars) are shown on the y-axis.

It may be taken therefrom that adoptive transfer of only 10³MCMV-specific cells markedly limited virus multiplication while 10⁴MCMV-specific cells nearly abolished virus replication in spleen. Nodifferences in protective capacity of CD8⁺ T cells generated followingRAE-1γMCMV and WT-MCMV infection were observed (see FIG. 4A). Recallresponse of memory CD8⁺ T cells was tested 6 months after the primaryinfection (see FIG. 4B and data not shown). The IE1/m123-specific andm164-specific CD8⁺ T cells in spleen, blood and tissue rapidly expandedupon challenge infection. Expansion peaked around day 6 after thechallenge resulting in T cell frequencies several orders of magnitudehigher than before the challenge in both RAE-1γMCMV and WT-MCMV infectedmice. Thus, although initial memory T cell pool was smaller inRAE-1γMCMV than in WT-MCMV infected mice, the size of the resulting Tcell pool after the challenge infection was similar in both groups ofmice. Collectively, these data indicate that despite tight innate immunecontrol, RAE-1γMCMV infection elicited a strong, enduring antiviralimmune response comparable to that following WT-MCMV infection.

Example 6 RAE-1γMCMV Immunization Protects Mice from Challenge Infection

To test whether the immune response induced by the RAE-1γMCMV infectionis sufficient to protect the host from challenge infection, adult BALB/cmice were footpad injected with 2×10⁵ PFU of RAE-1γMCMV or WT-MCMV 6months prior to lethal challenge with salivary gland derived MCMV (SGV).

The result is shown in FIG. 4C.

More particularly, FIG. 4C shows survival of naive mice (grey circles)and mice infected as described in FIG. 3 with RAE-1γMCMV (black circles)or WT-MCMV (white circles), respectively, which were i.p. challengedwith 2×10⁵ (left panel) or 5×10⁵ PFU (right panel) of SGV 6 mo p.i.Survival rates were monitored daily. One of two similar experiments isshown.

SGV is more virulent than the cell culture-derived MCMV and injectionwith only 10⁵ PFU of SGV results in multi-organ damage and highmortality (Trgovcich, J. et al., 2000, Arch Virol 145(12):2601-18).

It may be taken from FIG. 4C that RAE-1γ MCMV infection induceslong-term protective immunity. More particularly, while naïve micefailed to control the infection and succumbed to a dose of 2×10⁵ SGV (2LD50), all of the mice immunized with RAE-1γMCMV, similar to the micepreviously infected with WT-MCMV, survived the challenge (see FIG. 4C).Notably, mice immunized with RAE-1γMCMV resisted challenge infectionwith 5 LD50 of the SGV better than WT-MCMV infected mice, suggestingthat expression of NKG2D ligand provide an innate immune stimuli thatenhance the effectiveness of adaptive immune response. Taken together,immunization with RAE-1γMCMV induced an immune response that conferredprotection against lethal MCMV infection.

Example 7 Strong Attenuation In Vivo does not Prevent RAE-1γMCMV toEstablish Latent Infection and to Reactivate Upon Immunosuppression

The burden of latent viral DNA in a tissue predetermines the risk ofrecurrent CMV infection (Reddehase, M. J. et al., 2002, J Clin Virol 25Suppl 2:S23-36). The barely detectable DNA load of RAE-1γMCMV duringlatent infection could limit viral reactivation and subsequent recurrentvirus infection. However kinetics and phenotype of MCMV-specific T cellsobserved during latent infection were indicative of repeated antigenexposure. Therefore, the present inventor investigated the potential ofRAE-1γMCMV to reactivate from latency by combined depletion of NK cellsand T cell subsets in latently infected B-cell deficient μMT/μMT mice.

In this experimental system, the absence of antibodies facilitates virusmultiplication and dissemination after recurrence, which increases thesensitivity of virus detection (Jonjic, S. et al., 1994, supra).Following immunosuppression, recurrent infection occurred independentlyin different organs in 4 out of 6 (66%) RAE-1γMCMV infected mice and inall of the WT-MCMV infected mice, which may be taken from FIG. 5A.

More particularly, FIG. 5A shows the viral load of various organs, asdepicted on the x-axis, of μMT/μMT B cell-deficient mice latentlyinfected with RAE-1γMCMV (black circles) or WT-MCMV (white circles)which were depleted of CD4⁺, CD8⁺, and NK1.1⁺ cells by use of monoclonalantibody. Viral titers were determined by plaque assay 13 d afterimmunodepletion. Titers of individual mice (circles) and median values(horizontal bars) are shown as log₁₀ PFU per organ on the y-axis.Numbers indicate individual mice. DL means detection limit and isindicated by the dashed line.

It may be also taken therefrom that while in WT-MCMV infected micerecurrent infection first occurred in salivary glands favoring virusshedding, recurrence was not detected in salivary glands of anyRAE-1γMCMV infected mice. Thus, tight immune control of the RAE-1γMCMVduring primary infection did not prevent viral recurrence afterimmunosuppressive treatment altogether but altered incidence and sitesof recurrence.

Example 8 RAE-1γ Remains Intact During Latent RAE-1γMCMV Infection

Selective pressure from the immune system can result in emergence ofvirus mutants that escape from the immune control, even in herpesviruses with highly accurate mechanisms of genome replication (French,A. R. et al., 2004, Immunity 20(6):747-56; Voigt, V. et al., 2003, ProcNatl Acad Sci USA 100(23):13483-8). To address whether a strong immuneresponse can drive emergence of RAE-1γMCMV mutants that escape fromNKG2D-mediated immunesurveillance the present inventor preparedplaque-purified viruses from spleen and lung homogenates of B-celldeficient μMT/μMT mice with recurrent RAE-1γMCMV infection (see above).

The result is shown in FIG. 5B and FIG. 5C.

More particularly, FIG. 5B shows histograms of indicating the surfaceRAE-1γ expression of SVEC4-10 cells which were infected with theindicated recurrent plaque purified RAE-1γMCMV viruses and analyzed forthe surface RAE-1γ expression by FACS as described in FIG. 1B.

A total of 73 recurrent, plaque purified virus isolates (termedRAE-1γMCMVr1 to RAE-1γMCMVr73) were isolated from organ homogenates ofB-cell deficient μMT/μMT mice with recurrent RAE-1γMCMV infection. Inconnection therewith it will be understood that WTr as used herein is aWT-MCMV which was isolated as recurrent, plaque purified virus. Thewhite histogram indicates the surface expression of RAE-1γ afterWTr-MCMV infection.

FIG. 5C shows virus titer in spleen of untreated BALB/c mice (ø) orBALB/c mice treated with blocking anti-NKG2D antibody (αNKG2D) whichwere i.v. injected with 10⁵ PFU of WT-MCMV (white circles) or recurrentplaque purified RAE-1γMCMV (clone RAE-1γMCMVr5) (black circles) asindicated on the x-Axis. Viral titers were determined in spleen 3 d p.i.by plaque assay. Titers of individual mice (circles) and median values(horizontal bars) of a representative experiment are shown as log₁₀ PFUper spleen on the y-axis.

The 73 plaque purified isolates (termed RAE-1γMCMVr1 to RAE-1γMCMVr73)were tested for the expression of RAE-1γ and some of them were testedfor sensitivity to the NKG2D-mediated immune control in vivo. Infectionof SVEC4-10 cells with plaque purified isolates resulted in cell surfaceexpression of RAE-1γ detected by FACS analysis as may be taken from FIG.5B, and infection of BALB/c mice with a RAE-1γMCMVr isolate(RAE-1γMCMVr5) resulted in NKG2D-dependent attenuation of virusreplication similar to the attenuation of parental RAE-1γMCMV as may betaken from FIG. 5C. Finally, PCR amplification of RAE-1γ was performedand sequence analysis of PCR products did not reveal sequence variationin any of 30 RAE-1γMCMVr isolates tested (data not shown). These dataindicate that despite strong selective pressure imposed byNKG2D-dependent immune control mechanisms the RAE-1γ transgene encodedby RAE-1γMCMV remained intact.

Example 9 Control of RAE-1γMCMV in Mice Lacking the Receptor for Type IInterferons and after Haemoablative Irradiation

Type I interferons, also referred to herein as IFNs, play an importantrole in limiting MCMV replication during the early stage of infection.Consequently, mice lacking the receptor for type I IFNs, also referredto herein as IFNα/βR^(−/−), are 1,000-fold more susceptible to MCMVinfection than the parental mouse strain (Presti, R. M. et al., 1998, JExp Med 188(3):577-88). To test whether RAE-1γMCMV is efficientlycontrolled even in the severely immunodeficient host, IFNα/βR^(−/−) micewere i.p. injected with RAE-1γMCMV, WT-MCMV or Δm152 MCMV.

The result is shown in FIG. 6A.

More particularly, FIG. 6A shows the survival of IFNα/βR^(−/−) mice,also referred to herein as IFNa/bR ko mice, which were i.p. injectedwith 2×10⁵ PFU of RAE-1γMCMV (black circles), WT-MCMV (white circle) orΔm152-MCMV (grey circles) and survival rates of which were monitoreddaily. Combined results of two similar experiments are shown.

It may be taken therefrom that while most of the WT-MCMV and Δm152-MCMVinfected mice succumbed to the infection, i.e. 85% and 60%,respectively, the mortality rate of the RAE-1γMCMV infected animals wassignificantly lower (30%) (see FIG. 6A).

NK cells are more resistant to irradiation than other lymphoid cells(Ogasawara, K. et al., 2005, Nat Immunol 6(9):938-45; Erlach, K. C. etal., 2008, Med Microbiol Immunol 197(2):167-78) and RAE-1γMCMV isextremely sensitive to the NK cell control. The present inventorassessed whether residual NK cells, after hematoablative treatment, aresufficient to control RAE-1γMCMV infection. BALB/c mice werehematoablated using a sublethal dose (6 Gy) of total body γ-irradiation6 hours prior to footpad injection with 10⁵ PFU of RAE-1γMCMV or WT-MCMVand viral titers were compared on day 7 after infection.

The result is shown in FIG. 6B.

More specifically, FIG. 6B shows the viral load of spleen (left panel)and lungs (right panel) of BALB/c mice which were subjected to 6 Gytotal-body γ-irradiation 6 hours prior to f.p. injection with 10⁵ PFU ofRAE-1γMCMV (black circles) or WT-MCMV (white circles).

Were indicated mice were depleted for NK cells by anti-asialoGM1antibody (αGM1). Viral titers were determined 7 d p.i. by plaque assay.Titers of individual mice (circles) and median values (horizontal bars)are shown as log₁₀ PFU per organ on the y-axis. DL means detection limitand is indicated by the dashed line.

It may be taken therefrom that RAE-1γMCMV infection in hematoablatedmice resulted in significantly lower viral titers as compared to theWT-MCMV suggesting that residual NK cells are sufficient to restrainRAE-1γMCMV infection (see FIG. 6B).

Together these data argue that infection with the RAE-1γMCMV presents alow risk for disease, even in severely immunodeficient hosts.

Example 10 Maternal RAE-1γMCMV Immunization Protects Neonatal Mice fromMCMV Infection

Maternal preconception immunity to CMV provides substantial protectionagainst congenital infection (Fowler, K. B. et al., 2003, Jama289(8):1008-11; Boppana, S. B., and Britt, W. J., 1995, J Infect Dis171(5):1115-21; Boppana, S. B. et al., 2001, N Engl J Med344(18):1366-71). The presence of maternal antiviral antibodies isassociated with a decreased incidence of intrauterine transmission andbetter neurological outcomes in the setting of congenital infection. Therole of antibodies in the prevention of congenital infection has alsobeen emphasized in the guinea-pig CMV model (Schleiss, M. R., 2008, JClin Virol 41(3):224-30). Since the mouse hemoplacental barrier does notsupport MCMV transfer the present inventor established a model of i.p.neonatal MCMV infection whose pathogenesis closely resembles congenitalHCMV infection (Koontz, T. et al., 2008, J Exp Med 205(2):423-35). Totest whether the maternal antibody response induced by the RAE-1γMCMVimmunization can protect neonatal mice from MCMV infection, femaleBALB/c mice were injected with RAE-1γMCMV, WT-MCMV or mock infected twoweeks before mating. A number of neonates were sacrificed on the day ofbirth and tested for the presence of antiviral antibodies in serum,while the others were i.p. injected with 500 PFU of WT-MCMV and testedfor replicating virus in the tissue.

The results are shown in FIG. 7A and FIG. 7B.

More specifically, FIG. 7A, upper panel, shows the antiviral antibodytiter indicated as the Optical Intensity, OD, as determined by ELISA ofserum of female BALB/c mice which were i.v. injected with 2×10⁵ PFU ofRAE-1γMCMV (black squares) or WT-MCMV (white squares) or mock injected(grey squares) 2 weeks before mating. Antiviral antibody titers in theserum of said females (left panel) and in the serum of the neonates ofsaid females (right panel) were determined by ELISA 6 h post partum. Onthe x-axis the “-fold” factor of the dilution of the serum subjected toELISA is indicated. The lower panel is a illustrative scheme of theexperimental protocol.

FIG. 7B, upper panel shows an illustrative scheme of the experimentalprotocol, the lower panel shows a diagram indicating the viral load ofthe neonates of female BALB/c mice of FIG. 7A, wherein the females werei.v. injected with 2×10⁵ PFU of RAE-1γMCMV (black squares) or WT-MCMV(white squares) or mock injected (grey squares) 2 weeks before matingand wherein the neonates were i.p. injected with 500 PFU of WT-MCMV 6 hpost partum. Viral titers were determined in different organs 9 dayspost i.p. infection of neonates by plaque assay. Titers of individualmice (circles) and median values (horizontal bars) are shown as log₁₀PFU per gram neonate. DL means detection limit and is indicated by thedashed line.

It may be take therefrom that no antiviral antibodies were detected inthe serum of neonates of naive females. By contrast, antiviralantibodies were detected in serum of RAE-1γMCMV and WT-MCMV immunizedfemales and in serum of their neonates confirming passive placentaltransfer of antiviral antibodies (see FIG. 7A).

Whereas, MCMV infection in infected neonates of naive females resultedin disseminated virus replication, no replicating virus was detected invarious tissues at day 9 after the infection in neonates of RAE-1γMCMVimmunized females or in neonates of WT-MCMV immunized females (see FIG.7B).

Thus, immunization with recombinant RAE-1γMCMV induced a maternalantibody response that, upon placental transfer, limited virusdissemination and protected neonatal mice from MCMV infection.

Example 11 Generation and Characterization of Recombinant MCMVExpressing NKG2D Ligand RAE-1γ and Immunodominant CD8⁺ T Cell Epitope ofListeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellularpathogen which replicates in the cytoplasm and can spread from cell tocell without being exposed to extracellular environment. Immune responseto Listeria monocytogenes includes a complex network of cytokines andcells of innate and adaptive immunity (Unanue, E R, 1997, Immunol Rev.158:11-25). For the clearance of Listeria monocytogenes, interferon γ,also referred to herein as IFNγ, secreton during early days of infectionis required. IFNγ is provided by NK cells as well as by CD8⁺ T,therewith contributing to innate immune system in response to Listeriamonocytogenes.

RAE-1γMCMV expressing immunodominant CD8⁺ T cell epitope of Listeriamonocytogenes, also referred to herein as RAE-1γMCMVList, wasconstructed using orthotopic peptide swap method as described byLemmermann et al. (Lemmermann et al., 2010, supra) on RAE-1γMCMVbackbone where Dd-restricted antigenic m164 peptide ₁₆₇AGPPRYSRI₁₇₅(SEQ.ID.NO: 2) was swapped with the Kd-restricted listeriolysin O(LLO)-derived peptide ₉₁GYKDGNEYI₉₉ (SEQ. ID. NO: 3) (see FIG. 12A).

FIG. 12A shows a schematic illustration of the cloning process andgenome organization of RAE-1γMCMV and RAE-1γMCMVList. The HindIIIcleavage map of the MCMV genome is shown (MCMV) with the genomic regionencoding the m152 ORF below. In order to construct RAE-1γMCMV the m152ORF was replaced by an expression cassette comprising the HCMV majorimmediate early promoter (CMV-P), the RAE-1γ ORF (RAE-1γ) and the SV40polyadenylation signal sequence (pA).

The HindIII cleavage map of the RAE-1γMCMV genome is shown (RAE-1γMCMV)with the genomic region encoding the m164 ORF below. In RAE-1γMCMVListthe immunidominant m164 epitope (₁₆₇AGPPRYSRI₁₇₅; SEQ. ID. NO: 2) wasswapped with listeriolysin epitope (GYKDGNEYI; SEQ. ID. NO: 3).

RAE-1γMCMV backbone was constructed as described herein. In addition,MCMVList virus was constructed where Dd-restricted antigenic m164peptide ₁₆₇AGPPRYSRI₁₇₅ (SEQ.ID.NO: 2) was swapped with theKd-restricted listeriolysin O (LLO)-derived peptide ₉₁GYKDGNEYI₉₉ (SEQ.ID. NO: 3) in BAC-derived MCMV as a backbone.

FIG. 13 shows virus titers determined in supernatants of MEF which wereinfected with 0.1 PFU/cell of WT-MCMV (circles), MCMVList (squares) orRAE-1γMCMVList (triangles). Supernatants were harvested at time pointsp.i. as indicated on the x-axis and virus titers were determined byplaque assay. The virus titer is depicted on the y-axis as log₁₀ PFU permilliliter, ml.

It may be taken therefrom that listeriolysin epitope expression did notinterfere with growth of neither the WT-MCMV nor RAE-1γMCMV and bothMCMVList and RAE-1γMCMVList had replication kinetics comprable with theone of the WT-MCMV.

FIG. 12B shows an illustrative scheme of the experimental protocol ofexperiments applying Listeria monocytogenes challenge. Balb/c mice werevaccinated f.p. with 10⁵ PFU of WT-MCMV, MCMVList or RAE-1γMCMVList,respectively, at least three weeks prior to Listeria monocytogeneschallenge. Four days after challenge CFU in spleen and liver wasdetermined. Listeriolysin-specific CD8⁺ T cell response was measured onisolated splenocytes by stimulation with listeriolysin peptide andintracellular staining for IFNγ.

Example 12 Expression of NKG2D Ligand Enhances CD8⁺ T Cell ResponseAgainst Immunodominant Epitope Derived from Listeria monocytogenes

To test the effect of NKG2D ligand expression in response tolisteriolysin epitope, BALB/c mice were injected footpad (f.p) with2×10⁵ PFU of RAE-1γMCMV expressing listeriolysin epitope, also referredto herein as RAE-1γMCMVList, or WT-MCMV expressing listeriolysinepitope, also referred to herein as MCMVList. As already mentioned inExample 3 herein, MCMV expressing RAE-1γ was highly attenuated in vivo,which may also be taken from FIG. 14.

More particularly, FIG. 14 shows the results of two individualexperiments (left and right panel). Balb/c mice were f.p. infected with2×10⁵ PFU of MCMV expressing listeriolysin epitope, i.e. MCMVList (whitecircles), or MCMV expressing RAE-1γ ligand and listeriolysin epitope,i.e. RAE-1γMCMVList (black circles). Virus titers in salivary glandswere determined at various time points post infection by standard plaqueassay. Virus titers per organ for individual animals (circles) and asmedian values (bars) as log¹⁰ PFU per salivary gland on the y-axis, areshown. DL means detection limit and is indicated by the dashed line.

The kinetics of listeriolysin specific CD8⁺ T cell response was followedup to three months post infection. The results are shown in FIG. 15.BALB/c mice were infected with 10⁵ PFU f.p. of MCMVList or RAE-1γMCMVList. At indicated time points frequency of LLO specific CD8⁺ Tcells was determined. The results may be taken from FIG. 15A. Effectormemory (Tem, CD44⁺ CD62L⁻) and central memory (Tcm, CD44⁺ CD62L⁺)phenotype of listeriolysin specific CD8⁺ T cells was determined at day77 p.i. The results may be taken from FIG. 15B. Error bars show mean±SD.BALB/c mice were infected with 2×10⁵ PFU f.p. of MCMVList or RAE-1γMCMVList. At indicated time points splenocytes were isolated, stimulatedwith listeriolysin (FIG. 15C) or pp 89 peptide (FIG. 15D) andintracellulary stained for IFNγ production. Individual animals (circles)and median values are shown.

As may be taken therefrom the frequency of listeriolysin specific CD8⁺ Tcells was higher in mice immunized with RAE-1γMCMV expressinglisteriolysin compared with mice immunized with the virus expressinglisteriolysin epitope only. Moreover, listeriolysin specific CD8⁺ Tcells derived from both MCMVList and RAE-1γMCMVList infection retainedeffector memory phenotype. Altogether, this finding indicated that NKG2Dligand expressed in the context of MCMV enhances CD8⁺ T cell response toforeign epitope.

Example 13 Expression of NKG2D Ligand Dramatically Improves ProtectiveCapacity of MCMV Vector

The above results demonstrate that RAE-17 expressed in MCMV vectorconsiderably improves listeriolysin specific CD8⁺ T cell response. Nextthe present inventor tested how this correlates with protection ofvaccinated mice against challenge infection with Listeria monocytogenes.For that reason BALB/c mice were vaccinated with 10⁵ PFU f.p. ofWT-MCMV, MCMVList or RAE-1γMCMVList, or left unvaccinated. Three weekspost vaccination mice were challenged with 2×10⁵ CFU/mouse of Listeriamonocytogenes EGD.

The result is shown in FIG. 16A.

More particularly, FIG. 16 A shows colony-forming units of listeriamonocytogenes of Balb/c mice which were vaccinated f.p. with 10⁵ PFU ofWT-MCMV (dark grey bars), MCMVList (white bars), RAE-1γMCMVList (blackbars) or left uninfected (light grey bars, Ø). At ≧three weeks postvaccination all groups were challenged i.v. with Listeria monocytogenes,also referred to herein as LM, EGD strain serotype 1/2a. Challenge wasperformed with 3000 CFU of LM (low dose, left and right upper panel) and˜2×10⁴ CFU of LM (high dose, left and right lower panel). Four daysafter challenge CFU was determined in spleen (upper and lower leftpanel) and liver (upper and lower right panel). Results are presented asmean±SEM of five mice per vaccinated group; DL means detection limit andis indicated by the dashed line.

It may be taken therefrom that injection of Listeria monocytogenes intonaive mice resulted in high bacterial load in both spleen and liver (seeFIG. 16A). Mice vaccinated with MCMV expressing listeriolysin showedsignificantly lower load of bacteria, both in liver and spleen incomparison to naïve mice. However, mice vaccinated with RAE-1γMCMVListalmost cleared the bacteria from spleen (differences compared to naïvemice were ˜6 logs). Similar differences were detected in liver(difference of ˜5 logs). Therefore, NKG2D ligand expressed in context ofMCMV vector dramatically enhanced protective capacity mediated by CD8⁺ Tcells directed against single immunodominant epitope (see FIG. 16A).Similar results were obtained in three independent experiments. Inaddition, infection of mice with WT-MCMV failed to provide anyprotection against Listeria monocytogenes challenge, which may be alsotaken from FIG. 16A.

Additionally, four days post challenge paraffin embeded spleen sectionswere stained with aCD3 antibody.

The results are shown in FIG. 16B.

More particularly, FIG. 16B shows micrographs at a magnification of 40×of Paraffin-embeded spleen sections taken on day 4 post challenge andstained for CD3 expression of BALB/c mice infected with 1×10⁵ PFU/mouse[CAM: PFU?]f.p. of the indicated viruses or left uninfected. Three weeksp.i. mice were challenged with 1×10⁴ CFU/mouse of L. monocytogenes.

It may be taken therefrom that RAE-1γMCMVList immunization preserves aperiartheriolar lymphoid sheath (PALS) in L. monocytogenes challenge.

In addition, the efficacy of the RAE-1γMCMVList vaccine was alsoillustrated by the preservation of T cells in the periarteriolarlymphoid sheath of infected spleens, which are known to be depletedafter L. monocytogenes infection (Merrick, J. C., et al. 1997, Am JPathol 151(3): 785-92; Carrero, J. A. et al. 2004 J Immunol 172(8):4866-74)

Altogether, the herpesviral vector engineered to express the NKG2Dligand RAE-1γ generated a highly protective LLO-specific response thatwas able to efficiently cope with a L. monocytogenes challengeinfection. In vivo protective capacity correlated well with thefrequency of listeriolysin specific CD8⁺ T cells directed againstlisteriolysin epitope, as measured four days post challenge, which maybe taken from FIG. 17. This finding is more interesting bearing in mindthat the frequency of listeriolysin specific CD8⁺ T cells in mice beforechallenge was not so dramatically different.

More particularly, FIG. 17 shows the percentage of IFNγ⁺ CD8⁺ T cells ofsplenocytes from Balb/c mice which were f.p. vaccinated with 10⁵ PFU ofMCMVList (wt MCMVList, light grey bars), RAE-1γMCMVList (black bars) orleft unvaccinated (white bars, Ø). Three weeks post vaccination allgroups of mice were challenged with 2×10⁴ CFU of Listeria monocytogenes.Four days post challenge splenocytes were isolated, stimulated with 1listeriolysin peptide according to SEQ.ID.NO: 3 (left panel) orpp89-peptide according to SEQ. ID. NO: 1 (right panel), and stainedintracellularly for IFNγ production. The percentage of peptide specificIFNγ⁺ CD8⁺ T cells for five mice per group is shown. Results are shownas mean±SD and are representative of three individual experiments.

Example 14 Protective Capacity Against Listeria monocytogenes Challengein RAE-1γMCMVList Immunized Mice is CD8⁺ T Cell Dependant

In the light of the above described Examples, a person skilled in theart would expect that CD8⁺ T cells were responsible for protectionagainst challenge infection with Listeria monocytogenes. However, micevaccinated with either RAE-1γMCMVList or MCMVList were exposed toalthough immunodominant yet single epitope of this pathogen.

In connection therewith it is important to know that in the prior art itwas shown so far that previous infection with herpes viruses mayincrease the resistance of the host against unrelated bacterial or viralpathogens (Barton E S et al. 2007, Nature; 447(7142):326-9).

Therefore, four weeks after infection with RAE-1γMCMVList, MCMVList orWT-MCMV mice were challenged with 3000 CFU of Listeria monocytogeneswith and without depletion of CD8⁺ T cells.

The result is shown in FIG. 18.

More particularly, FIG. 18 shows colony-forming units of listeriamonocytogenes of.Balb/c mice which were f.p. vaccinated with 10⁵ PFU ofWT-MCMV (light grey bars), MCMVList (dark grey bars), RAE-1γMCMVList(black bars) or left unvaccinated (white bars). Four weeks postvaccination mice were challenged with 4×10³ CFU/mouse of Listeriamonocytogenes EGD. Prior to challenge mice were depleted for CD8⁺ Tcells or left undepleted. CFU in spleen and liver was determined fourdays post challenge., as indicated on the x-axis. Four days postchallenge CFU in liver (right panel) and spleen (left panel) wasdetermined. Results are shown as mean±SD for five mice per group. Colonyforming units are indicated as log₁₀CFU per organ on the y-axis.

It may be taken therefrom that the protective capacity of RAE-1γMCMVListis CD8⁺ T cell dependent. More particularly, RAE-1γMCMVList immunizedmice showed much lower bacterial load in spleen and liver, compared togroups of mice immunized with MCMVList, mice immunized with WT-MCMV ornaïve mice. These differences were practically abolished after depletionof CD8⁺ T cells confirming that these cells present dominant protectiveprinciple.

Pathohistological lesions in liver of infected mice were in accordancewith these results, which may be taken from FIG. 19.

More particularly, FIG. 19 shows micrographs (Magnification 200×) ofstainings of liver tissues from Balb/c mice which were leftunvaccinated, or vaccinated f.p. with 10⁵ PFU of WT-MCMV, MCMVList, andRAE-1γMCMVList. Four weeks after vaccination mice were challenged with3×10³ CFU of Listeria monocytogenes. Additionally, RAE-1γMCMVListvaccinated mice were depleted for CD8⁺ T cells prior to challenge. Fourdays post challenge staining of the liver tissues was performed. Normalunvaccinated mice were used as control (HE, x 200).

It may be taken therefrom that infection of naïve mice with Listeriamonocytogenes resulted in lesions throughout the liver. The lesions werecharacterized by multifocal inflammatory infiltrates with necrosis aswell as microvesicular vacuolation of the hepatocytes. Similarpathohistological lesions were observed in mice previously infected withWT-MCMV. Interestingly, massive multifocal lesions were also observed inmice immunized with MCMVList, despite the lower bacterial load in liversand spleens as compared to nonimmunised control groups. In contrast, theliver of mice immunized with RAE-1γMCMVList was practically of normalhistological appearance and only rare inflammatory foci were observed.Depletion of CD8⁺ T cells not only abolished protection but alsoresulted in multifocal inflammation and necrosis, similar to thoseobserved in naïve Listeria monocytogenes infected mice.

Altogether, these results showed that RAE-1γMCMV is a potent vector forCD8⁺ T cell based vaccine approach.

In Listeria monocytogenes infection, NK cells produce IFNγ as a resultof contact with infected bone marrow derived dendritic cells, alsoreferred to herein as BMDCs, as well as in a response to differentcytokines (Humann, J. and Lenz, L. L. 2010, J Immunol 184(9):5172-8).

As may be taken from FIG. 20, injection of Listeria monocytogenes intonaïve mice resulted in depletion of splenic NK cells.

More particularly, FIG. 20 shows the percentage of IFNγ+ NK cells andpercentage of total NK cells of splenocytes of Balb/c mice which weref.p. vaccinated with 10⁵ PFU of WT-MCMV (light grey bars),RAE-1γMCMVList (black bars) or left unvaccinated (white bars, Ø). Fourweeks post vaccination all groups of mice were challenged with 4×10³ CFUof Listeria monocytogenes. Four days post challenge splenocytes wereisolated and percentage of IFNγ producing NK cells (right panel) andpercentage of total NK cells (left panel) were determined. Naïve micenon-challenged with Listeria monocytogenes were used as a control(control, dotted bar). Results are shown as mean±SD and arerepresentative of three individual experiments.

Listeria monocytogenes challenge of naive and wt MCMV vaccinated miceresulted in dramatic reduction of splenic NK cells γ (FIG. 20, left). Incontrast, frequency of NK cells in RAE1gMCMVList vaccinated miceremained similar to uninfected control. Moreover, splenic NK cells inRAE-1γMCMVList vaccinated mice remained inactive after challengeinfection. However, remaining NK cells in naive and wt MCMV immunizedmice after challenge infection showed activation phenotype asdemonstrated by expression of CD69 (not shown) and IFNγ production (FIG.20, right). Thus, the results indicate that efficient CD8⁺ T cellresponse in RAE-1γMCMVList not only preserved NK cell frequency butthere was also no need for their activation, most likely becausebacteria fail to reach splenic area and induce NK cell response

Example 15 Enhanced CD8⁺ T Cell Response in Mice Infected withRAE-1γMCMVList Correlates with Preserved DCs

It is well established that systemic MCMV infection results in depletionof splenic conventional dendritic cells (cDCs). Here the presentinventor tested the impact of WT-MCMV and RAE-1γMCMVList on thefrequency of cDC subsets in spleen.

The result is shown in FIG. 21.

More particularly, FIG. 21 shows CDa⁺DCs (Cd11c^(hi) CD8a⁺, left panel)and CD11bDCs (Cd11c^(hi) CD8a⁻, right panel) of Balb/c mice which werei.v. infected with 2×10⁵ PFU of WT-MCMV (light grey bars),RAE-1γMCMVList (black bars) or left uninfected (white bars). Onindicated days post infection splenocytes were isolated and stained forthe frequency of CD8a⁺DCs (Cd11c^(hi) CD8a⁺, left panel) and CD11bDCs(Cd11c^(hi) CD8a⁻, right panel) within CD3⁻ CD19⁻ MHCI+ population.Results are shown as mean±SD for three mice per group and arerepresentative of two individual experiments. The number of CDa⁺DCs(Cd11c^(hi) CD8a⁺, left panel) and CD11bDCs (Cd11c^(hi) CD8a⁻, rightpanel) cells, respectively, is depicted as the 10⁵th part of totalnumber (#×10⁵) on the y-axis.

More precisely, mice were infected i.v. with indicated viruses or leftuninfected and splenic cDCs were isolated using collagenase D digestionand analyzed for CD3⁻ CD19⁻ MHCII⁺ CD11c^(hi) CD8α expression. It may betaken from FIG. 21 that on day 1.5 p.i. the frequency of CD8αDCs(CD11c^(hi) CD8α⁺ DCs) in WT-MCMV infected mice was already lower thanin control non infected mice (see FIG. 21, left panel). The decrease inCD8αDCs cells was even more pronounced on day 3.5 p.i. However, in miceinfected with RAE-1γMCMVList the frequency of CD8αDCs was not affected,or was even slightly increased at later time point. The frequency ofCD11bDCs (CD11c^(hi) CD8α⁻ DCs) was reduced after infection withWT-MCMV, but also after infection with RAE-1γMCMVList (see FIG. 21,right panel).

Yet, the loss of CD11b DC subset was less pronounced in RAE-1γMCMVListinfected mice when compared to WT-MCMV infected (see FIG. 21).

Next the present inventor tested frequency of plasmacytoid dendriticcells (pDCs) and serum level of interferon alfa (IFNα). IFNα is known topromote NK cell cytotoxicity (Biron, C A. et al., 1999, Annu RevImmunol. 17:189-220), but can also act immunosuppressive when producedin high concentrations.

The result is shown in FIG. 22.

More particularly, FIG. 22 shows the percentage of pDC (left panel) andthe concentration of IFNα of Balb/c mice which were i.v. infected with2×10⁵ PFU of WT-MCMV (light grey bars), RAE-1γMCMVList (black bars) orleft uninfected (white bars) on time points post infection as indicatedon the x-axis. Splenocytes were isolated and stained for B220⁺ PDCA-1⁺phenotype (left panel) or the concentration of the IFNα in the sera ofthe infected mice was determined (right panel). Results are shown asmean±SD for three mice per group.

It may be taken therefrom that there was no significant difference inthe frequency of pDCs during infection with both WT-MCMV andRAE-1γMCMVList. Interestingly, however, the serum level of IFNα at day1.5 was significantly higher in mice infected with WT-MCMV as comparedto RAE-1γMCMVList infected mice (see FIG. 22).

The above results indicate that early events after infection might bedecisive for the differences observed in protective capacity ofRAE-1γMCMVList. Therefore the present inventor also tested early CD8⁺ Tcell response upon infection with either WT-MCMV, MCMVList orRAE-1γMCMVList.

The result is shown in FIG. 23.

More particularly, FIG. 23 shows the total number of CD8⁺ T cells (leftpanel), effector memory CD8⁺ T cells (middle panel) and virus specificCD8⁺ T cells (right panel) of Balb/c mice which were i.v. infected with2×10⁵ PFU of WT-MCMV (light grey bars; light grey circles),RAE-1γMCMVList (black bars; black circles) or left uninfected (whitebars; white circles) on days post infection as indicated on the x-axis.Results are shown as mean±SD for three mice per group and arerepresentative of two individual experiments.

It may be taken therefrom that a higher frequency of effector memory aswell as virus specific CD8⁺ T cells is detected upon RAE-1γ infection.

Example 16 Generation of HA-Containing Recombinant Viruses

The further test vector capacity of MCMV expressing NKG2D ligand Rae1γ,the present inventor inserted influenza virus PR8 hemagglutinin, alsoreferred to herein as HA, in WT-MCMV and RAE-1γMCMV genome, resulting inMCMV-HA and RAE-1γMCMV-HA, respectively.

The construction of recombinant plasmids comprising HA expressioncassette, and recombinant HA-full (RAE-1γMCMVHA) or HA-headless(RAE-1γMCMVHAheadless) RAE-1γMCMV is schematically shown in FIG. 24A,FIG. 24B, FIG. 24C, FIG. 24D and FIG. 24E.

More particularly, FIG. 24 A to E show schematic illustrations of thegenome organization and cloning process of HA-full and HA-headlessRAE-1γMCMV.

FIG. 24 A shows the HindIII cleavage map of the MCMV genome at the top,with the genomic region encoding the m152 ORF and m157 ORF below. Them152 ORF was preplaced by an expression cassette (bottom) comprising theRAE-1γ ORF, the HCMV major immediate early prompter (CMV-P) and the SV40polyadenylation signal sequence (pA). The m157 ORF was replaced by anexpression cassette (bottom) comprising the HA-full ORF, the HCMV majorimmediate early prompter (CMV-P) and the SV40 polyadenylation signalsequence (pA).

FIG. 24 B shows the HindIII cleavage map of the MCMV genome at the top,with the genomic region encoding the m152 ORF and m157 ORF below. Them152 ORF was preplaced by an expression cassette (bottom) comprising theRAE-1γ ORF, the HCMV major immediate early prompter (CMV-P) and the SV40polyadenylation signal sequence (pA). The m157 ORF was replaced by anexpression cassette (bottom) comprising the HA-headless ORF, the HCMVmajor immediate early prompter (CMV-P) and the SV40 polyadenylationsignal sequence (pA).

FIG. 24 C shows a schematic illustration of the construction of WT- andRAE-1γ MCMV expressing PR8 influenza hemagglutinin full form comprisingthe steps of:

-   I. PCR amplification of expression plasmid with HCMV MIEP and KanR;    pGL3 plasmid provided by Invitrogen (left panel) and PCR    amplification of PR8 HA-ORF provided by Peter Stäheli,    Universitätsklinikum Freiburg, Germany;-   II. Clone PR8 HA-ORF into expression plasmid (blunt-end ligation)-   III. BglII restriction of plasmid with HA-expression cassette-   IV. Homologous recombination with MCMV-BAC, replacing the m157 ORF,    whereby the KanR cassette will subsequently be removed using Sce-I    endonuclease. FIG. 24 D shows a schematic illustration of the    construction of WT- and RAE-1γ    MCMV expressing PR8 influenza hemagglutinin headless form comprising    the steps of-   I. PCR amplification of plasmid with HCMV MIEP, KanR and PR8 HA    full-ORF;-   II. Ligation of PCR amplified DNA fragment;-   III. BglII restriction of plasmid with HA-headless-expression    cassette; and-   IV. Homologous recombination with MCMV-BAC, replacing the m157 ORF,    whereby the KanR cassette will subsequently be removed using Sce-I    endonuclease.

FIG. 24 E shows schematic illustrations of the genome organization ofMCMV mutants expressing PR8-HA full form (upper panel) and PR8-HAheadless form (lower panel) with GGGG linker (see also Steel J. et al.,2010, MBio. 18; 1(1)). To generate the HA full and headless MCMVmutants, an ORF encoding V5-tagged HA full was first cloned into pGL3plasmid together with a Tischer-modified kanamycin resistance gene(kanR), which was inserted further downstream. The HA-expressioncassette was linearized by restriction at BglII unique sites outsidem157 homology regions. The linearized fragment was integrated into theBAC by red-α, -β, -γ-mediated recombination, thereby replacing them157ORF. The kanR cassette was subsequently excised with Sce-Iendonuclease encoded by GS1783 bacterail cells (provided by Tischer B.K.).

It will also be understood that hmIEP referres to the major immediateearly promoter of HCMV; HA1 refers to the H-2 Kd-Balb/c restrictedpeptide HA533-541; HA2 refers to the H-2 Kd-Balb/c restricted peptideHA533-541; In connection therewith it will be understood that HA1 is asubunit of HA. One domain of HA1 is a globular head that is deleted inthe case of headless construct; HA2 is stalk subunit of HA, which ishighly conserved among different influenza strains. Accordingly, bothHA1 as well as HA2 contain peptide HA533-541. SV40pA refers to the SV40polyadenylation signal sequence and AmpR refers to an ampicillinresistance gene.

It may be taken from the above that Hemagglutinin was inserted in aplace of m157, a gene coding for protein which is directly recognized byNK cell receptor Ly49H (Arase et al., 2002, supra). The generatedviruses were designated as MCMV-Δm157-HA, also referred to hereinasMCMV-HA. and RAE-1γMCMV-Δm157-HA, also referred to herein asRae1γMCMV-HA, respectively.

In connection therewith it is important to note that previous studiesshowed that engagement of Ly49H with m157 upon MCMV infection leads toactivation of NK cells and subsequently better control of infection inLy49H positive mice. When m157 deletion mutant MCMV is used, C57BL/6mice lost the ability to control the virus and infection results in highvirus titers in visceral organs and salivary glands (Bubic, I. et al.,2004, J Virol. 78(14):7536-44).

The major problem in designing efficient influenza vaccine is mutationof viral genes encoding immunodominant proteins. The stalk region ofhemagglutinin is conserved among different strains and therefore ispotentially a good candidate for generation of cross-protective immuneresponse (Steel J. et al., 2010, supra). Therefore, in addition torecombinant MCMV expressing entire HA, the present inventor has alsogenerated above mentioned viruses expressing headless hemagglutinininserted in a place of m157, namely RAE-1γMCMVHA, RAE-1γMCMVHAheadless,MCMV HA, MCMV HA. Since H2b restricted immunodominant epitope₁₁₄YPYDVPDYA₁₂₂ (SEQ.ID.NO: 38) is positioned in a head ofhemagglutinin, the present inventor has additionally inserted ovalbuminimmunodominant H2b restricted peptide ₂₅₇SIINFEKL₂₆₄(SEQ.ID.NO: 10) inthe stalk region of hemagglutinin to allow the present inventorfollowing CD8⁺ T cell response to well described foreign epitope.

It is important to understand that the H2b-B6 mouse restricted peptideHA114-122 (YPYDVPDYA)-(SEQ.ID.NO: 38) is not present in case ofHA-“headless” mutant.

The H-2 Kd-Balb/c restricted peptide HA533-541 (N-IYSTVASSL-C)(SEQ.ID.NO: 37) is present in both headless and full length forms of HAPR8.;

Recombinant plasmids were constructed according to establishedprocedures, and enzyme reactions were performed as recommended by themanufacturers. Throughout, the fidelity of PCR-based cloning steps wasverified by sequencing (GATC, Freiburg, Germany).

Growth kinetics of MCMVΔm157-HA and REA-1γMCMVΔm157-HA were compared toWT-MCMV.

The result is shown in FIG. 25.

More particularly, FIG. 25 shows virus titers determined in supernatantsof MEF which were infected with 0.1 PFU per cell of WT-MCMV (circles),Rae1γMCMV Δm157-HA (triangles) MCMV Δm157-HA (diamonds). Supernatantswere harvested at time points p.i. as indicated on the x-axis and virustiters were determined by plaque assay. The virus titer is depicted onthe y-axis as log₁₀ PFU per milliliter, ml. Each sample analysis wasperformed in duplicates.

It can be taken therefrom that insertion of hemagglutinin into the MCMVgenome had no effect on the replication kinetics of the recombinantviruses (see FIG. 25), in other words insertion of hemagglutinin doesnot affect virus growth in vitro.

Example 17 Efficient CD8⁺ T Cell Response to Influenza HA afterInfection with RAE-1γMCMV-HA

Recombinant WT-MCMV and RAE-1γMCMV with IE1; m123/SIINFEKL orm164/SIINFEKL-peptide swap were constructed as described in Example 1above.

FIG. 26A shows a schematic illustration of the construction of MCMVexpressing SIINFEKL peptide comprising the steps of:

-   I. PCR amplification of KanR cassette and introduction of SIINFEKL    (light grey block) and BAC homology regions, here homology to m123,    (black blocks); and-   II. Homologous recombination with WT MCMV-BAC, replacing the    respective position of the BAC according to homology regions, here    m123 ORF, whereby the KanR cassette will subsequently be removed    using Flp recombinase;-   III. Optionally PCR amplification of RAE-1γ expression cassette,    comprising major immediate early promoter of HCMV (MIEP), RAE-1γ ORF    and Kanamycin Resistance gene (KanR), thereby introduction of BAC    homology regions (dark grey blocks);-   IV. Homologous recombination with MCMV-SIINFEKL BAC as provided by    steps I. and II. according to Lemmermann et al. (Lemmermann et al.,    2010, supra), thereby replacing the respective position of the BAC    according to the homology regions, here the m152 ORF, whereby KanR    cassette will subsequently be removed using Flp recombinase;

FIG. 26B shows the HindIII cleavage map of the MCMV genome at the top,with the genomic region spanning genes m150 through m165 expanded belowto demonstrate the position of the ORFs of interest. The m152 ORF waspreplaced by an expression cassette (bottom) comprising the RAE-1γ ORF,the HCMV major immediate early prompter (CMV-P) and the SV40polyadenylation signal sequence (pA). The SIINFEKL-peptide (SEQ.ID.NO:10) was swapped in the m164 ORF as denoted in bold, resulting inRAE-1γMCMVm164SIINFEKL.

FIG. 26C shows the HindIII cleavage map of the MCMV genome at the top,with the genomic region spanning genes m121 to m124, and region fromm150 to m153 expanded below to demonstrate the position of the ORFs ofinterest. The m152 ORF was preplaced by an expression cassette (bottom)comprising the RAE-1γ ORF, the HCMV major immediate early prompter(CMV-P) and the SV40 polyadenylation signal sequence (pA). The SIINFEKLpeptide (SEQ.ID.NO: 10) was swapped in the m123 ORF resulting inRAE-1γMCMVm123SIINFEKL.

To test whether hemagglutinin inserted in MCMV genome induces specificCD8⁺ T cell response, and how the NKG2D ligand expression influencesthis response, the present inventor has infected C57BL6 mice withMCMV-Δm157-HA or RAE-1γMCMV-Δm157-HA.

The result is shown in FIG. 27A and FIG. 27B.

More particularly, FIG. 27A shows the percentage of IFNγ+ CD8+ T cellsas a result of peptide-stimulation of splenocytes from C57BL/6 micewhich were infected with 2×10⁵ PFU f.p. of MCMV-HA (white circles) andRAE-1γMCMVHA (black circles). The splenocytes were stimulated withindicated peptides, i.e. Hemagglutinin (HA)-peptide HA533-541(N-IYSTVASSL-C) (SEQ.ID.NO: 37) (left panel) and Ovalbumin(SIINFEKL)-peptide (SEQ.ID.NO: 10) (right panel). Hemagglutinin (HA) andOvalbumin (SIINFEKL)-specific CD8+ T cell response was followed for 60days. Splenocytes were isolated at time points indicated on the x-axisand stimulated with indicates peptides. IFNγ⁺ CD8⁺ T cell response, as aresult of indicated peptides stimulation, is shown. Bars representmedian values.

FIG. 27B shows the percentage of IFNγ+ CD8+ T cells as a result ofpeptide-stimulation of splenocytes from C57BL/6 mice which were infectedwith 2×10⁵ PFU f.p. of MCMV-HA (white circles) and RAE-1γMCMVHA (blackcircles). The splenocytes were stimulated with indicated peptides, i.e.M45-peptide (SEQ.ID.NO: 31) (upper left panel), IE3-peptide (SEQ.ID.NO:29) (upper right panel), m139-peptide (SEQ.ID.NO: 30) (lower left panel)and M38-peptide (SEQ.ID.NO: 32) (lower right panel). Peptide-specificCD8+ T cell response was followed for 60 days. Splenocytes were isolatedat time points indicated on the x-axis and stimulated with indicatespeptides. IFNγ⁺ CD8⁺ T cell response, as a result of indicated peptidesstimulation, is shown. Bars represent median values.

It may be taken therefrom that the hemagglutinin and SIINFEKL(SEQ.ID.NO: 10)-specific CD8⁺ T cell response was followed up to 60 dayspost infection (see FIG. 27A). Both, MCMV-Δm157-HA andRAE-1γMCMV-Δm157-HA infected mice generated and retained hemagglutininand SIINFEKL (SEQ.ID.NO: 10)-specific CD8⁺T cell response duringanalyzed period of time with similar frequency. There was no significantdifference in the frequency of CD8⁺ T cells following MCMV-Δm157-HA orRAE-1γMCMV-Δm157-HA. Therefore CD8⁺T cell response to MCMV specificimmunodominant epitopes obtained after RAE-1γMCMV-Δm157-HA infection wasequal or even higher than after MCMV-Δm157-HA infection (see FIG. 27).

It was further assessed whether RAE-1γMCMV-Δm157-HA is attenuated inC57BL/6 mice.

The result is shown in FIG. 28.

More particularly, FIG. 28 shows the viral load of lungs (upper andlower right panel), liver (upper and lower middle panel) and spleen(upper and lower right panel) of C57BL/6 mice which were infected with10⁵ PFU of WT-MCMV (light grey circles), Δm157FRT (triangles) (Bubic, I.et al, 2004, supra), MCMVHA (white circles) and RAE-1γMCMVHA (blackcircles).

Virus titers were determined in depicted organs at 4 days post infection(upper panels), and 8 day post infection (lower panels).

Virus titers for individual animals (circles and triangles) and medianvalues (bars) are shown as log₁₀ PFU per organ on the y-axis. Detectionlimit is indicated by the horizontal line.

It may be taken therefrom that virus expressing NKG2D ligand areattenuated in C57BL/6 mice (see FIG. 28).

Gazit et al. (Gazit, R. et al., 2006, Nat. Immunol. 7(5):517-23)demonstrated that Ncr1 receptor expressed on NK cells is essential forelimination of influenza virus infected cells in vivo by directrecognition of viral HA protein. Therefore, the present inventor hastested whether Ncr1 would have effect on generation of hemagglutininspecific CD8⁺ T cell response in MCMV-Δm157-HAorRAE-1γ-MCMV-Δm157-HAinfecion.

The result is shown in FIG. 29.

More particularly, FIG. 29 shows the percentage of IFNγ⁺ CD8⁺ T cells ofsplenocytes of C57BL/6 and C57BL/6 NCR^(gfp/gfp) mice, respectively, asindicated on the x-axis, which were infected with 2×10⁵ PFU f.p. of MCMVΔm157-HA (white circles) or Rae1γMCMV Δm157-HA (black circles).Splenocytes were isolated at 8 days p.i. (left panel), SIINFEKL-peptide(SEQ.ID.NO: 10) (middle panel) and M45-peptide (SEQ.ID.NO: 31) (rightpanel). The percentage of IFNγ⁺ CD8⁺ T cells of splenocytes is indicateon the y-axis. Bars represent median values.

It may be taken therefrom that RAE-1γMCMV-Δm157-HA generates efficientCD8⁺ T cell response regardless of the role of Ncr1 in vivo. Nodifference in HA specific CD8⁺T cell response was observed at day 8 p.i.in C57BL/6 mice when compared to MCMV-Δm157-HAor RAE-1γMCMV-Δm157-HAinfection in NCR^(−/−) mice (see FIG. 29).

Example 18 ULBP2 Expressing HCMV Recombinant

To test NKG2D impact on virus attenuation in human MCMV model, the sameprinciple as in RAE-1γMCMV was applied and recombinant HCMV mutants weregenerated expressing UL16 binding protein 2 (ULBP2, also known as RAET1H), an NKG2D ligand, in place of UL16 gene which codes for the proteindownregulating ULBP2.

FIG. 30A and FIG. 30 B schematically show the construction ofrecombinant ULBP2-HCMV virus mutants.

More particularly, FIGS. 30 A and B show schematic illustrations of thegenome organization and cloning process of ULBP2 expressing HCMV.

FIG. 30A shows a schematic illustration of the construction ofrecombinant ULBP2-HCMV virus comprising the steps of

-   I. PCR amplification of ULBP2-ORF from ULBP2-encoding plasmid    provided by Open Biosystems, subsequently subjected to enzyme    restriction with KpnI and BamHI;-   II. Clone into expression plasmid with MCMV MIEP and KanR applying    restriction enzyme digestion and ligation;-   III. Generation of PCR fragment for recombination, wherein homology    regions to target site of recombination, here to UL16, is depicted    as dark grey boxes; and-   IV. Homologous recombination with HCMV TB40E-BAC, thereby replacing    the UL16 ORF, whereby KanR cassette will subsequently be removed    using Flp recombinase.

FIG. 30B shows the HindIII cleavage map of the HCMV AD169 genome at thetop, with the genomic region spanning genes UL15A to UL17 expanded belowto demonstrate the position of the ORF UL16. The UL16 ORF was replacedby an expression cassette (bottom) comprising the ULBP2 OR, the MCMVmajor immediate early promoter (CMV-P) and the SV40 polyadenyltaionsignal sequence (pA).

Next ULBP2 expression was detected in TB40E-infected fibroblasts byimmunoblotting.

DNA of two BAC clones (#39, #41) generated as described above weretransfected into human fibroblasts as described by Borst et al. (Borst,E. M. et al., 2007, supra). For comparison DNA of the parental BAC ofthe TB40E strain (Sinzger, C. et al., 2008, J Gen Virol., 89(Pt2):359-68) was transfected. Infectious virus was reconstituted andfibroblasts were incubated until complete cytopathic effect occurred.Cells were harvested, lysed and proteins of the lysates were separatedby SDS-PAGE, blotted and probed with an ULBP-2 specific antibody (R&DCat. No. AF1298, 1:1000) followed by incubation with an HRP-anti-goatantibody (1:1000) and visualization of the signal with ECL substrate.

The results are shown in FIG. 30C.

More particularly, FIG. 30 C shows a Western blot analysis, wherein theimmunoblot has been exposed to a film for detection of signal for ashort exposure time (left panel) and for a long exposure time (rightpanel). As controls lysates from non-infected human fibroblast (HFFn.i), lysates from fibroblast infected with the BAC-cloned CMV strainRVHB15 (AD169-derived) {Borst et al. 1999; Hobom et al., 2000}, asmentioned above fibroblasts infected with the virus reconstituted fromthe parental TB40E BAC, as well as lysates from MCMV infected murineembryonic fibroblasts (MCMV on MEF) were used.

As may be taken therefrom substantial amounts of ULBP2 were detected inTB40-ULBP2 #39 and #41 infected cells, whereas basically no ULBP2expression were detected in noninfected human fibroblasts and smallamounts of ULBP2 expression were detected in RVHB15 and TB40E-infectedfibroblasts.

Furthermore the Surface expression of ULBP2 after infection of HFF withHCMV-ULBP2 and control virus was assessed.

The results are shown in FIG. 30D and FIG. 30E.

More particularly, FIG. 30D shows FACS analysis of ULBP-2 expression.Human foreskin fibroblasts (HFF) were infected with 1 PFU of HCMV TB40strain or HCMV TB40 expressing ULBP-2, or left uninfected. 24 hours postinfection cells were tested for ULBP-2 expression.

Furthermore the results of NK cell assay using HFF infected withHCMV-ULBP2, HCMV TB40 and uninfected HFF as targets are shown in FIG.30E.

FIG. 30E shows that HCMV TB40 ULBP2 expressing virus promotescytotoxicity of NK cells. Freshly isolated human NK cells were used aseffectors. Human foreskin fibroblasts as target cells were infected with1 PFU/cell of HCMV TB40 strain or HCMV TB40 expressing ULBP-2, or leftuninfected. 24 hours post infection, target cells were mixed in threedifferent ratios 10:1, 5:1 and 2.5:1 with NK cells isolated form healthyblood donor to determine the effect of ULBP-2 on NK cell cytotoxicityusing the Promega CytoTox assay. LDH release from necrotic cells wasdetermined by assaying culture media samples. Mean and SD values areshown. Circles (uninfected HFFs), squares (HCMV strain TB40 infectedHFFs), triangles (HCMV TB40 strain lacking UL16 gene expressingULBP-2).]

Example 19 General Protocol for Vaccinating a Human Subject Using aRecombinant HCMV Expressing ULBP2

The recombinant HCMV used in accordance with this general protocol is aHCMV as described in Example 1 expressing ULBP2. The recombinant HCMVexpressing ULBP2 is for use in a method of vaccinating the subjectagainst HCMV and thus for use in a method for eliciting an immuneresponse against HCMV.

Inclusion and Exclusion Criteria

A subject is admitted to participate in the vaccination trial if thehuman subject is a male or female human between 22 and 60 years of ageat the time of enrollment into the trial, if the informed consent isobtained from the subject before vaccination, if the subject is healthy,as determined by a questionnaire concerning the medical history of thesubject and clinical examination, and if the subject is tested to beseronegative for HCMV.

A subject is excluded from participation in the vaccination trial if thesubject is tested to be seropositive for HCMV, if the subject is testedto be pregnant, if the subject is or has been undergoing drug therapy orvaccination within 30 days preceding the vaccination trial, if thesubject was previously vaccinated against HCMV, if the subject has orhad frequent recurrent herpes simplex infections, if the subject has orhad any immunodeficiency, if the subject has or had a Hepatitis Binfection or hepatitis C infection, if the subject has or had a medicalhistory of allergic disease or reactions, if the subject has or had anymajor chronic illness including diabetes mellitus, if the subject has orhad a medical history of any neurologic disease, if the subject issuffering from a malignancy, if the subject has an acute disease at thetime of participation in the trial and/or if the subject has a medicalhistory of administration of immunoglobulins or blood products withinthree months preceding the enrollment into the trial or if the subjecthas a history of chronic alcohol consumption or drug abuse.

Administration

HCMV expressing ULBP2 is used to inoculate the subject by a subcutaneousinjection with 50 infectious units as determined by titration onpermissive human fibroblast cells. Alternatively, the recombinant HCMVexpressing ULBP2 is administered by oral inoculation with 250 infectiousunits. A placebo group is inoculated with the pharmaceuticallyacceptable carrier used in connection with the subcutaneous injectionand oral inoculation of the HCMV expressing ULBP2.

A blood sample is taken from the patient before vaccination and a secondblood sample is taken between day 28 and day 32 post vaccination.

Infection of the host is documented by comparison of the analysis of theblood sample taken before vaccination and the analysis of the bloodsample taken between day 28 and day 32 post vaccination, whereby theblood samples are analyzed for

-   -   (1) serological evidence of infection with HCMV,    -   (2) serological evidence of antibody response to HCMV, and    -   (3) evidence for the presence of specific CD8⁺T cells to HCMV.

Efficacy

To determine the efficacy of the recombinant HCMV expressing ULBP2 thekinetics and magnitude of the CMV-specific immune response is assessedin the healthy HCMV-sero-negative participants compared to the placebogroup.

The efficacy of the HCMV-specific immune response is assessed asfollows.

a) Determination of systemic HCMV infection is determined as follows:

At day 0, as well as 1, 2, 6, 12 and 24 months post vaccination a urinesample, a blood sample and a saliva sample is taken from theparticipants and analyzed for HCMV using PCR.

b) Determination of antibody titers directed against HCMV.

antibody titers directed against HCMV-specific proteins determined byneutralization and enzyme-linked immunosorbent assay (ELISA) and/or byWestern blot assays in samples taken at 1, 6 and 12 months followingvaccination.

2 months following vaccination, a 15 ml of blood sample is taken fromthe subjects and peripheral blood mononuclear cells (PBMC) are recoveredby standard technologies. PBMC are stimulated in standard assays fordetection of the proliferation of CD8⁺ T lymphocytes and CD4⁺ Tlymphocytes in response to

(1) HCMV antigens (viral lysate).

In addition, in patients in which appropriate HLA haplotypes can bedefined, CD8+ T lymphocyte responses are assayed using ELISPOT and flowcytometry following ex-vivo stimulation of mononuclear cells with theheterologous antigen and CMV antigens pp 65, IE-1 and gB.

Protection

The vaccination is considered successful if positive serologicalresponses to the HCMV-specific antigens are detected. More specifically,a serological response is considered positive if a more than 4-fold risein antibody levels is detected and/or if viral nucleic acids aredetected in the urine The emphasis is on assessment of HCMV-specific CD8T cell priming and maintenance following vaccination with HCMV ULBP2.Regarding the CD8 T cell response the vaccination is consideredsuccessful if virus-specific CD8 T cell response against HLA haplotypematched HCMV infected human foreskin fibroblasts is similar to orexceeding the one in HCMV seropositive control subjects, as assessed byproliferation capacity and frequency of HCMV-specific CD8 T cells, aswell as their capacity for IFNg production.

The results of these studies are quantified and compared to identicalstudies carried out at 6, 12, and 24 months following vaccination.

Surrogates of vaccine efficacy include antibody levels reactive withHCMV proteins and more importantly CD8+ T lymphocyte responses for HCMVantigens.

Example 20 Treatment of a Human Subject Using a Recombinant HCMVExpressing ULBP2

A 25 year old female human subject is treated in accordance with thegeneral protocol of Example 19, whereby the recombinant HCMV expressesULBP2. The recombinant HCMV expressing ULBP2, as described in Example 1herein, is administered subcutaneously in a phosphate-buffered salinesolution containing 50 infectious units of the recombinant HCMV.

A blood sample is taken from the subject at day 30 post vaccination andthe titer of antibody against HCMV-specific protein is determined byELISA. A significant antibody immune response against HCMV-specificprotein is detected.

Furthermore, a blood sample is taken from the subject two months postvaccination and proliferation of both CD8⁺ T lymphocytes and CD4⁺ Tlymphocytes is observed in response to (1) HCMV antigens afterstimulation with lysate of HLA matched infected cells or (2) in responseto virus-specific peptide epitopes, as indicated in the Example 19.

Example 21 General Protocol for Vaccinating a Human Subject Using aRecombinant HCMV Expressing a Heterologous Antigen

The recombinant HCMV used in accordance with this general protocol is aHCMV as described in Example 1 expressing ULBP2 and a heterologousantigen against which an immune response is to be elicited in a humansubject. Such heterologous antigen is one as described in the instantspecification and includes more specifically influenza hemagglutininprotein, antigen 85A or HIV-1 gag protein. The recombinant HCMVexpressing influenza hemagglutinin protein is for use in a method ofvaccinating the subject against influenza and thus for use in a methodof eliciting an immune response against influenza. The recombinant HCMVexpressing antigen 85A of mycobacterium tuberculosis is for use in amethod of vaccinating the subject against mycobacterium tuberculosis andthus for use in a method of eliciting an immune response againstmycobacterium tuberculosis. The recombinant HCMV expressing HIV-1 gagprotein of HIV is for use in a method of vaccinating the subject againstHIV and thus for use in a method of eliciting an immune response againstHIV.

Inclusion and Exclusion Criteria

A subject is admitted to participate in the vaccination trial if thehuman subject is a male or female human between 22 and 60 years of ageat the time of enrollment into the trial, if the informed consent isobtained from the subject before vaccination, if the subject is healthy,as determined by a questionnaire concerning the medical history of thesubject and clinical examination, and if the subject is tested to beseronegative for HCMV.

A subject is excluded from participation in the vaccination trial if thesubject is tested to be seropositive for HCMV, if the subject is testedto be pregnant, if the subject is or has been undergoing drug therapy orvaccination within 30 days preceding the vaccination trial, if thesubject was previously vaccinated against HCMV, if the subject has orhad frequent recurrent herpes simplex infections, if the subject has orhad any immunodeficiency, if the subject has or had a Hepatitis Binfection or hepatitis C infection, if the subject has or had a medicalhistory of allergic disease or reactions, if the subject has or had anymajor chronic illness including diabetes mellitus, if the subject has orhad a medical history of any neurologic disease, if the subject issuffering from a malignancy, if the subject has an acute disease at thetime of participation in the trial and/or if the subject has a medicalhistory of administration of immunoglobulins or blood products withinthree months preceding the enrollment into the trial or if the subjecthas a history of chronic alcohol consumption or drug abuse.

Administration

HCMV expressing ULBP2 and the heterologous antigen is used to inoculatethe subject by a subcutaneous injection with 50 infectious units asdetermined by titration on permissive human fibroblast cells.Alternatively, the recombinant HCMV expressing ULBP2 and theheterologous antigen is administered by oral inoculation with 250infectious units. A placebo group is inoculated with thepharmaceutically acceptable carrier used in connection with thesubcutaneous injection and oral inoculation of the HCMV expressing ULBP2and the heterologous antigen.

A blood sample is taken from the patient before vaccination and a secondblood sample is taken between day 28 and day 32 post vaccination.

Infection of the host is documented by comparison of the analysis of theblood sample taken before vaccination and the analysis of the bloodsample taken between day 28 and day 32 post vaccination, wherebytheblood samples are analyzed for

(1) serological evidence of infection with HCMV,

(2) serological evidence of antibody response to the heterologousantigen, and

(3) evidence for the presence of specific CD8⁺ T cells to theheterologous antigen.

Efficacy

To determine the efficacy of the recombinant HCMV expressing ULBP2 andthe heterologous antigen protein the kinetics and magnitude of theCMV-specific and antigen-specific immune response is assessed in thehealthy HCMV-sero-negative participants compared to the placebo group.

The efficacy of the HCMV-specific immune response and the specificimmune response against the heterologous antigen are assessed asfollows.

a) Determination of systemic HCMV infection is determined as follows:

At day 0, as well as 1, 2, 6, 12 and 24 months post vaccination a urinesample, a blood sample and a saliva sample is taken from theparticipants and analyzed for HCMV using PCR.

b) Determination of antibody titers directed against HCMV-specificproteins and of antibody titers against the heterologous antigen.

1, 6 and 12 months following vaccination antibody titers directedagainst HCMV-specific proteins and against the heterologous antigendetermined by neutralization and enzyme-linked immunosorbent assay(ELISA) and/or by Western blot assays in samples taken at 1, 6 and 12months following vaccination.

2 months following vaccination, a 15 ml of blood sample is taken fromthe subjects and peripheral blood mononuclear cells (PBMC) are recoveredby standard technologies. PBMC are stimulated in standard assays fordetection of the proliferation of CD8⁺ T lymphocytes and CD4⁺ Tlymphocytes in response to

(1) HCMV antigens (viral lysate), and

(2) the heterologous antigen.

In addition, in patients in which appropriate HLA haplotypes can bedefined, CD8⁺ T lymphocyte responses are assayed using flow cytometryfollowing ex-vivo stimulation of mononuclear cells with the heterologousantigen and CMV antigens pp 65, IE-1 and gB.

Protection

The vaccination is considered successful if positive serologicalresponses to the heterologous antigen and optionally also theHCMV-specific antigens are detected. More specifically, a serologicalresponse is considered positive if a more than 4-fold rise in antibodylevels is detected and/or if viral nucleic acids are detected in theurine.

The results of these studies are quantified and compared to identicalstudies carried out at 6, 12, and 24 months following vaccination.

Surrogates of vaccine efficacy include antibody levels reactive with theheterologous antigen and more importantly CD8+ T lymphocyte responsesfor the heterologous antigen.

Example 22 Treatment of a Human Subject Using a Recombinant HCMVExpressing Influenza Hemagglutinin Protein

A 25 year old female human subject is treated in accordance with thegeneral protocol of Example 21, whereby the recombinant HCMV expressesinfluenza hemagglutinin protein. The recombinant HCMV expressinginfluenza hemagglutinin protein, as described in Example 1 herein, isadministered subcutaneously in a phosphate-buffered saline solutioncontaining 50 infectious units of the recombinant HCMV.

A blood sample is taken from the subject at day 30 post vaccination andthe titer of antibody against HCMV-specific protein and against theinfluenza hemagglutinin protein is determined by ELISA. A significantantibody immune response against both HCMV-specific protein and againstthe influenza hemagglutinin protein is detected.

Furthermore, a blood sample is taken from the subject two months postvaccination and proliferation of both CD8⁺ T lymphocytes and CD4⁺ Tlymphocytes in response to (1) HCMV antigens (viral lysate), (2)influenza hemagglutinin protein and (3) ULBP2 is observed.

The HCMV-ULBP2 HA vaccine elicits cross-reactive anti-HA2 antibodies andanti-HA2 CD8⁺ T cells superior than the response detected in humansubjects during natural influenza infection or subjects vaccinated withconventional vaccines against seasonal influenza. Quality of T cellresponse is determined by ELISPOT and flow cytometry on PBMC isolatedprior to vaccination and following vaccination during the outbreak ofseasonal influenza epidemics or after vaccination with conventionalseasonal influenza vaccine. T cell response induced by HCMV-ULBP2expressing HA are considered successful if superior than specific T cellresponse in control subjects, i.e. individuals after vaccination withconventional seasonal influenza vaccine and subjects after naturalinfluenza infection. The subjects vaccinated with HCMV-ULBP2 HA headlessvaccine develop serum neutralizing anti-HA2 antibodies against theepitopes which are not induced neither by infection with HCMV-ULBP2expressing full-length HA nor during the natural influenza infection.Such “unnatural” response to unmasked conserved HA epitope is beneficialover conventional vaccines with regard to cross-protective capacityagainst various influenza virus strains.

Example 23 Treatment of a Human Subject Using a Recombinant HCMVExpressing HIV-1 gag

A 25 year old female human subject is treated in accordance with thegeneral protocol of Example 21, whereby the recombinant HCMV expressesHIV-1 gag. The recombinant HCMV is administered subcutaneously in aphosphate-buffered saline solution containing 50 infectious units of therecombinant HCMV.

A blood sample is taken from the subject at day 30 post vaccination andthe titer of antibody against HCMV-specific protein and against theHIV-1 gag is determined by ELISA. A significant antibody immune responseagainst both HCMV-specific protein and against the HIV-1 gag isdetected.

Furthermore, a blood sample is taken from the subject two months postvaccination and proliferation of both CD8⁺ T lymphocytes and CD4⁺ Tlymphocytes in response to (1) HCMV antigens (viral lysate), (2) HIV-1gag and (3) ULBP2 is observed.

For subjects vaccinated with HCMV-ULBP2 vaccine expressing HIV-1 gagantigen the successful T cell priming of gag-specific CD4+ and CD8+T-cell responses is judged by ELISPOT and flow cytometry on PBMCisolated prior to vaccination and two months following vaccination. Theobtained result is compared to the placebo group and the group of elitecontrollers of HIV infection. The vaccination is considered successfulif the specific T cell responses are the same or exceeding the responsesin the group of elite controllers of HIV infection.

Example 24 Treatment of a Human Subject Using a Recombinant HCMVExpressing Antigen 85A

A 25 year old female human subject is treated in accordance with thegeneral protocol of Example 21, whereby the recombinant HCMV expressesAntigen 85A. The recombinant HCMV is administered subcutaneously in aphosphate-buffered saline solution containing 50 infectious units of therecombinant HCMV.

A blood sample is taken from the subject at day 30 post vaccination andthe titer of antibody against HCMV-specific protein and against theAntigen 85A is determined by ELISA. A significant antibody immuneresponse against both HCMV-specific protein and against the Antigen 85Ais detected.

Furthermore, a blood sample is taken from the subject two months postvaccination and proliferation of both CD8⁺ T lymphocytes and CD4⁺ Tlymphocytes in response to (1) HCMV antigens (viral lysate), (2) Antigen85A and (3) ULBP2 is observed.

The vaccination with HCMV-ULBP2 expressing mTB Antigen 85 (Ag85) isconsidered successfull in the terms of eliciting similar or betterimmune response against M. Tuberculosis in the group vaccinated withHCMV-ULBP expressing Ag85 as compared to samples of patients withcontrolled mTB infections (i.e. patients exposed and infected butsymptom free, without requirement for chemotherapy). Recall response insubjects receiving HCMV ULBP2 expressing Ag85 vaccine is consideredsuccessful if intradermal skin test response to mTB proteins is equal orbetter than in the unvaccinated group.

Example 25 RAE-1γMCMVList Provides Long-Term Protection AgainstChallenge Infection

The endurance of protective immunity is a prerequisite for an efficientprotection against re-infection. To assess whether the vaccination withan MCMV vector expressing RAE-1γ provides a long-lasting protectionagainst L. monocytogenes, BALB/c mice were f.p. infected with 1×10⁵ PFUof WT-MCMV, MCMVList, RAE-1γMCMVList, respectively, or left uninfected.60 days post-vaccination groups of MCMVList or RAE-1γMCMVList infected,or uninfected BALB/c mice were challenged with 2×10⁴ CFU/mouse of L.monocytogenes.

The result is shown in FIG. 32.

More particularly, FIG. 32 A shows the percentage of survival by day 4and body weight loss (mean±s.e.m.) on day 3 post-challenge (n=9-12 pergroup) (upper panel). The bacterial load in spleen and liver inindividual animals (circles) and median values (horizontal bars) ofsurvived mice on day 4 post-challenge are shown. DL means detectionlimit, and t indicates the death of a mouse.

It may be taken therefrom that all unvaccinated mice succumbed toinfection by day four, which was accompanied by a dramatic weight lossby day three post-challenge. Similar to this was the case of miceinfected with wt MCMV, although few mice survived the infection by dayfour, confirming that persistent MCMV infection might have a protectiveeffect against intracellular bacteria (Barton, E. S., 2007 supra.

Vaccination with MCMVList provided a substantial protection of theimmunized mice, but these mice exhibited significant weight loss. AllRAE-1γMCMVList-vaccinated mice survived the infection with minimalweight loss by day four post-challenge, once again suggesting thatRAE-1γMCMVList vaccination retains a long-lived and protective memoryCD8⁺ T cell response.

Mice that survived four days post challenge were sacrificed and thebacterial loads in liver and spleen were determined. Bacterial load inspleens of RAE-1γMCMVList vaccinated mice was below the limit ofdetection and significantly lower in livers when compared to WT-MCMV andMCMVList vaccinated mice.

In order to test whether RAE1γMCMVList-vaccinated mice could resist thechallenge with higher doses of L. monocytogenes, vaccinated mice werechallenged with 4×10⁴ CFU/mouse and monitored for survival.

The result is shown in FIG. 32 B.

More particularly, FIG. 32 B shows the survivorship curve of mice whichwere vaccinated as described above and which were injected with 4×10⁴CFU/mouse of L. monocytogenes. The survival rate was followed (n=9 pergroup).

It may be taken therefrom that while all unvaccinated mice succumb toinfection and MCMVList vaccinated mice began to die by day 4 postchallenge, all RAE-1γMCMVList vaccinated mice survived the challenge.

The efficient and long-lasting protective capacity of the LLO-specificCD8⁺ T cell response in mice vaccinated with RAE-1γMCMVList wasconfirmed by assessing the LLO-specific CD8+ T cell-mediatedcytotoxicity in vivo.

The result is shown in FIG. 32 C.

More particularly, FIG. 32 C shows the percentage oflisteriolysinpeptide-specific killing in mice which were infected f.p.with 1×10⁵ PFU of MCMVList or RAE-1γMCMVList (n=3-4 per group). After 2and 11 months p.i., mice were injected with an equal ratio ofunstimulated and LLO-peptide stimulated CSFE-stained splenocytes. TheMean±s.e.m. is shown.

It may be taken therefrom that listeriolysin-specific killing wassignificantly higher up to 11 months post-vaccination in mice vaccinatedwith RAE-1γMCMVList compared to those vaccinated with MCMVList.

Example 26 RAE-1γ Expression by MCMV is Crucial for its Vaccine VectorCapacity

To test the possibility that an enhanced LLO-specific CD8⁺ T cellresponse in RAE-1γMCMVList-infected mice is entirely a consequence ofthe deletion of the m152 gene, whose protein product (m152/gp40) notonly down-regulates the expression of MHC I molecules but also RAE-1, avirus expressing the LLO epitope on the backbone of the m152-deficientvirus (Δm152MCMVList) was constructed.

BALB/c mice were infected with 2×10⁵ PFU/mouse i.v. of the indicatedviruses. On day 8 post infection viral titer in lungs was determined.

The result is shown in FIG. 33 A.

More particularly, in FIG. 33 A the viral load in lungs of individualanimals is shown as circles and the median value of a group is shown ashorizontal line.

It may be taken therefrom, that Δm152MCMVList showed attenuated growthin vivo, but the attenuation was much stronger with RAE-1γMCMVList.

BALB/c mice were infected with 1×10⁵ f.p. of the indicated viruses. Thefrequency of LLO-specific CD8⁺ T cells was determined on day 7 p.i.

The result is shown in FIG. 33 B.

More particularly, in FIG. 33 B median values are shown as horizontallines.

It may be taken therefrom that the LLO-specific CD8⁺ T cell response inmice infected with Δm152MCMVList was similar to that induced byMCMVList, confirming that an enhanced CD8⁺ T cell response inRAE-1γMCMVList-infected mice was predominantly a consequence of ectopicexpression of RAE-1γ rather than the deletion of immune evasion genem152.

In connection therewith it has to be acknowledged that co-stimulationvia NKG2D plays an important role in shaping the CD8⁺ T cell response(Markiewicz, M. A., et al2005, “J Immunol 175(5): 2825-33; Barber, A.and C. L. Sentman, 2011, Blood 117(24): 6571-81). This function may beof central importance for the success of RAE-1γMCMV as a vaccine vector,since MCMV down-regulates co-stimulatory molecules on antigen-presentingcells similarly to HCMV (Loewendorf, A., et al. 2004 J Virol 78(23):13062-71; Mintern, J. D., et al. 2006, J Immunol 177(12): 8422-31;Arens, R., et al. 2011, J Virol 85(1): 390-6) and RAE-1-NKG2Dinteraction may rescue the co-stimulation signals during T cell priming.

BALB/c mice were infected with 2×10⁵ i.v. of WT-MCMV; MCMVList orRAE-1γMCMVList, respectively. One day before infection and on days 2 and4 p.i. mice were treated with NKG2D blocking antibody

The result is shown in FIG. 34.

More particularly, FIG. 34 A shows the virus titer in spleen which wasdetermined on day 3 p.i. Individual animals are shown as circles andmedian values are shown as horizontal lines, and FIG. 34 B showsabsolute number of LLO-specific CD8⁺ T cells which were determined ondays 3.5 and 6.5 p.i. Mean±SEM is shown.

It may be taken therefrom that NKG2D stimulation by MCMV is importantfor the listeriolysin-specific CD8+ T cells generation.

More particularly, blocking of NKG2D by specific monoclonal antibodiessignificantly reduced the control of RAE-1γMCMVList at day 3post-infection (see FIG. 34A). However, this treatment only modestlylowered the CD8⁺ T cell response (see FIG. 34B). An alternativeexplanation for the modest effect of NKG2D blocking on the CD8⁺ responsecould simply be a higher antigenic viral load caused by NKG2D blockingwhich, on its own, may facilitate the CD8 response via cross-priming(Lemmermann, N. A., V. Bohm, et al. (2011). “In vivo impact ofcytomegalovirus evasion of CD8⁺ T-cell immunity: facts and thoughtsbased on murine models.” Virus Res 157(2): 161-74).

BALB/c mice were infected with 2×10⁵ i.v. of MCMVList, RAE-1γMCMVList orleft untreated (naïve). On days 4 and 8 post infection mice wereinjected with 2 mg of BrdU/mouse and sacrified 2 h later.

The frequency of BrdU⁺ total CD8⁺ T cells, the frequency of LLO-specificCD8⁺ T cells and the frequency of BrdU⁺ LLO-specific CD8⁺ T cells wasdetermined.

The result is shown in FIG. 35.

More particularly, FIG. 35 A shows the frequency of BrdU⁺ total CD8⁺ Tcells, FIG. 35 B shows the frequency of LLO-specific CD8⁺ T cells andFIG. 35 C shows the BrdU⁺ LLO-specific CD8⁺ T cells. Mean±SEM is shown.

It may be taken therefrom that RAE-1γ expression promoteslisteriolysin-specific CD8⁺ T cells priming. The preserved frequency ofDCs during the early days of infection corresponds to the enhancedpriming of CD8⁺ T cells in RAE-1γMCMVList-infected mice, which isillustrated by the higher frequencies and proliferation capacity ofLLO-specific CD8⁺ T cells.

Altogether, the above data demonstrates that the expression of the NKG2Dligand RAE-1γ by CMV vectors promotes the priming of an epitope-specificCD8⁺ T cell response.

Example 27 RAE-1γMCMV is an Efficient Vaccine Vector in C57BL/6 Mice

To exclude the possibility that the robustness of the CD8⁺ T cellresponse after infection with RAE-1γMCMVList vector was restricted to asingle MHC I haplotype, a recombinant RAE-1γMCMV and wt MCMV expressingthe H-2K^(b) restricted peptide SIINFEKL instead of m164 epitope wereconstructed as described in Example 1 above, referred to herein asRAE-1γMCMV-SIINFEKL and MCMV-SIINFEKL, respectively (Rotzschke, O., etal. 1991), Eur J Immunol 21(11): 2891-4).

Growth kinetics of MCMV-SIINFEKL and REA-1γMCMV-SIINFEKL were comparedto WT-MCMV.

More particularly, MEF cells were infected with wt MCMV, MCMV-SIINFEKLor RAE-1γMCMV-SIINFEKL at 0.1 PFU per cell. Supernatants were harvestedat indicated time p.i. and virus titers were determined by plaque assay.

The result is shown in FIG. 36.

It may be taken therefrom that the expression of SIINFEKL peptide didnot impair the virus growth in vitro.

C57BL/6 (H-2^(b)) mice were infected with the viruses expressingSIINFEKL via footpad or intravenous route and kinetics of SIINNFEKL- andvirus-specific CD8⁺ T cell response was followed up to three months postinfection.

The results are shown in FIGS. 37 and 38.

More particularly, FIG. 37 shows the kinetics of SIINFEKL specific CD8⁺T cell response after infection of C57BL/6 mice with 10⁵ PFU/mouse f.p.of the indicated viruses.

SIINFEKL- and MCMV-specific CD8⁺ T cell response has been followed for89 days. IFNγ⁺ CD8⁺ T cell response, as a result of indicated peptidesstimulation, is shown for individual animals as triangles. Median valuesare shown as bars.

FIG. 38 shows the kinetics of SIINFEKL specific CD8⁺ T cell responseafter infection of C57BL/6 mice with 10⁵ PFU/mouse i.v. of the indicatedviruses SIINFEKL- and MCMV-specific CD8⁺ T cell response has beenfollowed for 89 days. IFNγ⁺ CD8⁺ T cell response, as a result ofindicated peptides stimulation, is shown for individual animals astriangles. Median values are shown as bars.

It may be taken therefrom that in agreement with the results obtained inBALB/c mice, the infection of C57BL/6 mice with RAE-1γMCMV-SIINFEKLresulted in a stronger CD8⁺ T cell response to SIINFEKL compared to thevirus expressing SIINFEKL only. The CD8⁺ T cell response to some MCMVimmunodominant epitopes was also higher in RAE-1γMCMV-SIINFEKLinfection, which confirms the previously published data (Slavuljica, I.,et al. 2010, J Clin Invest 120(12): 4532-45).

To assess the protective capacity of RAE-1γ expressing MCMV vector inC57BL/6 mice, mice were immunized with 10⁵ PFU/mouse f.p. with virusesexpressing SIINFEKL either with or without RAE-1γ co-expression and werechallenged with low and high dose of Listeria expressing ovalbumin(OVA-Listeria) three weeks later.

Four days post challenge bacterial load in spleen and liver wasdetermined.

The result is shown in FIG. 39.

More particularly, FIG. 39 shows the viral lowed in spleen (left panel)and liver (right panel) of C57BL/6 mice which were infected with 10⁵PFU/mouse f.p of the indicated viruses, or left uninfected. Three weekspost infection mice were challenged with either 10⁴ CFU/mouse (low dose)or 5×10⁴ CFU/mouse (high dose). Four days post challenge bacterial loadin spleen and liver was determined, shown as mean±SEM.

It may be taken therefrom that contrary to a modest protective capacityin BALB/c mice immunized with MCMVList, the protective response ofC57BL/6 mice immunized with MCMV-SIINFEKL was much stronger, to thepoint that the beneficial effect of RAE-1γMCMV-SIINFEKL vaccination washardly visible. However, the beneficial effect of RAE-1γ expressionbecame evident after a challenge infection of mice with a higher dose ofOVA-Listeria.

Previous studies showed that cross-presentation plays a dominant role inthe priming of CD8⁺ T cells during MCMV infection. The present inventorproposes that MCMV expressing RAE-1γ favors direct priming due todramatically lowered antigenic load which should reduce thecross-priming capacity of such a virus. To test the role of directpresentation in infection with the virus expressing RAE-1γ 3 d micewhich are defective in TLR3, TLR7 and TLR9 signaling and unable tocross-present foreign antigens (Tabeta, K., et al. 2006, Nat Immunol7(2): 156-64).

C57BL/6 and 3 d mice were infected with 2×10⁵ PFU/mouse i.p. of eitherMCMV-SIINFEKL or RAE-1γMCMV-SIINFEKL, or left uninfected. Seven dayspost infection the frequency of SIINFEKL-specific as well asMCMV-specific CD8⁺ T cells was determined 7 days later, and viral titerin lungs was determined.

The result is shown in FIG. 40.

More particularly, FIG. 40 A shows the frequency of SIINFEKLtetramer-specific CD8⁺ T cells (left panel) and M45 tetramer-specificCD8⁺ T cells of individual animals as triangles and median values areshown as bars.

FIG. 40 B shows the viral titer in lungs.

It may be taken therefrom that RAE-1γ expression by MCMV vector promotesdirect priming of vectored antigen-specific CD8+ T cells.

Although both viruses induced an epitope-specific CD8⁺ T cell response,the response induced by RAE-1γMCMV-SIINFEKL was slightly better than theone in mice infected with MCMV-SIINFEKL. Thus, the absence ofcross-presentation does not compromise the robust CD8⁺ T cell responseto the viruses expressing RAE-1. In addition, RAE-1γMCMV-SIINFEKL wasreadily controled in MCMV sensitive 3 d mice when compared toMCMV-SIINFEKL.

Example 28 Influenza Challenge Experiments

C57BL/6 mice were immunized with 2×10⁵ PFU/mouse f.p. of the Δm157 MCMV,MCMV-HA or RAE-1γMCMV-HA, or left non-immunized. Three weeks postimmunization mice were intranasally challenged with either high or lowdose (100 HU) of human influenza virus A/Puerto Rico/8/34 H1N1, alsoreferred to herein as A/PR8. A/PR8 was generated as previously described(Achdout, H., et al. 2003 J Immunol 171(2): 915-23). For the low dose,40 hemagglutinin units (HU) of PR8 virus were used. Mice were monitoreddaily for survival and weight loss.

The result is shown in FIG. 41.

More particularly, in FIG. 41 A the survival of mice immunized withΔm157 MCMV, MCMV-HA or RAE-1γMCMV-HA, or non-immunized mice is shownover time.

In FIG. 41 B the weight loss of mice immunized with Δm157 MCMV, MCMV-HAor RAE-1γMCMV-HA, or non-immunized mice is shown over time. Sigificaldifference was calculated using unpaired Student's t-test, n=5mice/group.

Example 29 Vector Capacity of RAE-1γMCMV-CD8 Epitope of RSV

BALB/c mice were infected with 10⁵ PFU/mouse f.p. of MCMV-SYI orRAE-1γ-MCMV-SYI or left uninfected and SYIGSINNI-specific CD8⁺ T cellresponse has been followed.

Generation of Recombinant Viruses Expressing Respiratory Syncytial Virus(RSV) M2-Derived Peptide SYIGSINNI

Recombinant plasmids were constructed according to establishedprocedures, and enzyme reactions were performed as recommended by themanufacturers. Throughout, the fidelity of PCR-based cloning steps wasverified by sequencing (GATC, Freiburg, Germany).

(i) Design of Insert Containing Peptide Swap.

Primers were constructed in a way to replace the Dd-restricted antigenicm164 peptide 167-AGPPRYSRI-175 with the Kd-restricted M2-derived peptide82-SYIGSINNI-90.

The primers were m164 SYI fw(5′-cgcccgctgccacgatggcctggttgttgacggcccagaagatgttgttgatcgagccgatgtagctgtcagcgccccaGCCAGTGTTACAACCAATTAACC-3′) (lower case letters represent homology region tom164 ORF in MCMV genome, bold underline letters represent introducedepitope SYIGSINNI, bold italic letters homology regions between primersand capital letters represent homology to Tischer kanamycin cassette)and m164 SYI rv(5′-gccgttcggaaaggactactgtcggacgtggggcgctgacagctacatcggctcgatcaacaacatcTAGGGATAACAGGGTAATCGAT-3′) (lower case letters represent homology region to m164 ORFin MCMV genome, bold underline letters represent introduced epitopeSYIGSINNI, bold italic letters homology regions between primers andcapital letters represent homology to Tischer kanamycin cassette). PCRwas performed with the following cycler conditions: an initial step for2 min at 98° C. for activation of HighFidelity Phusion DNA polymerase(New England BioLabs) was followed by 30 cycles of 10 s at 98° C., 10 sat 60° C., and 60 s at 72° C. As DNA template plasmid pEP-SaphAI, a kindgift from K. Tischer was used.

(ii) BAC Mutagenesis.

For the construction of recombinant mutants en passant mutagenesis; atwo step markerless red recombination system was utilized, as describedby Tischer, B. K. et al, 2010, supra. Mutagenesis of full-length mCMVbacterial artificial chromosome (BAC) and dm152-RAE1γ mCMV BAC wasperformed in Escherichia coli strain DH10B, whereas excision of theselection marker was performed in Escherichia coli strain GS1783.

The result is shown in FIG. 42.

More particularly, FIG. 42 shows the kinetics of SYIGSINNI-specific CD8⁺T cell response in BALB/c mice which were infected with 10⁵ PFU/mousef.p. of the indicated viruses or left uninfected. SYIGSINNI-specificCD8⁺ T cell response has been followed at indicated time points.Splenocytes were stimulated with SYIGSINNI peptide and frequency ofIFNγ⁺ CD8⁺ T cells is shown as mean±SEM.

It may be taken therefrom that the frequency of_SYIGSINNI-specific CD8⁺T cells is higher in mice infected with MCMV-SYI or RAE-1γ-MCMV-SYIcompared to uninfected mice.

The frequency of SYIGSINNI-specific CD8⁺ T cells increases from day 7 today 14 p.i. in mice infected with MCMV-SYI or RAE-1γ-MCMV-SYI.

The frequency of SYIGSINNI-specific CD8⁺ T cells in RAE-1γ-MCMV-SYIinfected mice is higher compared to MCMV-SYI infected mice.

The features of the present invention disclosed in the specification,the claims, the sequence listing and/or the drawings may both separatelyand in any combination thereof be material for realizing the inventionin various forms thereof.

What is claimed is:
 1. A beta-herpesvirus, preferably a recombinantbeta-herpesvirus, wherein the beta-herpesvirus comprises at least oneheterologous nucleic acid, wherein the at least one heterologous nucleicacid comprises a gene encoding a cellular ligand.
 2. Thebeta-herpesvirus according to claim 1, wherein the cellular ligand iscapable of binding a receptor for the cellular ligand wherein thereceptor for the cellular ligand is present on the surface of at leastone immune cell, and wherein the at least one immune cell is selectedfrom the group consisting of NK cells, γδ T cells and activated CD8⁺ Tcells.
 3. The beta-herpesvirus according to claim 1, wherein thecellular ligand is an NKG2D ligand.
 4. The beta-herpesvirus according toclaim 3, wherein the NKG2D ligand is a human NKG2D ligand is selectedfrom the group consisting of UL16 binding proteins and MHCclass-1-related protein.
 5. The beta-herpesvirus according to claim 4,wherein the UL16 binding protein is selected from the group consistingof ULBP2, ULPB1, ULBP3, ULBP4, ULBP5 and ULBP6.
 6. The beta-herpesvirusaccording to claim 4, wherein the MHC class-1-related protein isselected from the group consisting of MICA and MICB.
 7. Thebeta-herpesvirus according to claim 1, wherein the beta-herpesvirus issuitable for inducing an immune response against a beta-herpesvirus,wherein the immune response comprises neutralizing antibodies againstbeta-herpesvirus and/or CD4⁺ T-cells directed against epitopes ofbeta-herpesvirus and/or CD8⁺ T-cells directed against epitopes ofbeta-herpesvirus.
 8. The beta-herpesvirus according to claim 1, whereinthe beta-herpesvirus is human cytomegalovirus.
 9. The beta-herpesvirusof claim 1, wherein the beta-herpesvirus is deficient in at least onegene product encoded by an immune modulatory gene.
 10. Thebeta-herpesvirus according to claim 9, wherein the at least one immunemodulatory gene is selected from the group consisting of UL16 and UL142.11. The beta-herpesvirus according to claim 9, wherein thebeta-herpesvirus is deficient in one or more additional gene product(s)each encoded by an additional immune modulatory gene.
 12. Thebeta-herpesvirus according to claim 11, wherein the at least oneadditional gene product encoded by the additional immune modulatory geneis a gene product regulating NK cell response encoded by an immunemodulatory gene selected from the group consisting of UL16, UL18, UL40,UL142, m152, m155, m145 and m138.
 13. The beta-herpesvirus of claim 11,wherein the at least one additional gene product encoded by theadditional immune modulatory gene is a gene product regulating MHC classI presentation, wherein the gene product regulating MHC class Ipresentation is a gene product encoded by an immune modulatory geneselected from the group consisting of US6, US3, US2 and US11.
 14. Thebeta-herpesvirus according to claim 1, wherein the beta-herpesviruscomprises the deletion of at least one miRNA.
 15. The beta-herpesvirusaccording to claim 1, wherein the beta-herpesvirus is deficient in atleast one gene product encoded by a gene regulating viral replication,wherein the gene regulating viral replication is selected from the groupconsisting of IE1, pp 71 and pp65.
 16. The beta-herpesvirus according toclaim 1, wherein the beta-herpesvirus is deficient in at least one geneproduct encoded by an essential gene, wherein the essential gene isselected from the group consisting of UL32, UL34, UL37.1, UL44, UL46,UL48, UL48, UL49, UL50, UL51, UL52, UL53, UL54, UL55, UL56, UL57, UL60,UL70, UL71, UL73, UL75, UL76, UL77, UL79, UL80, UL84, UL85, UL86, UL87,UL89.1, UL90, UL91, UL92, UL93, UL94, UL95, UL96, UL98, UL99, UL100,UL102, UL104, UL105, UL115 and UL122.
 17. The beta-herpesvirus accordingto claim 1, wherein the beta-herpesvirus is deficient in at least oneglycoprotein.
 18. The beta-herpesvirus according to claim 1, wherein thebeta-herpesvirus encodes at least one additional heterologous nucleicacid.
 19. The beta-herpesvirus according to claim 18, wherein the atleast one additional heterologous nucleic acid is a functional nucleicacid selected from the group consisting of antisense molecules,ribozymes and RNA interference mediating nucleic acids or wherein the atleast one additional heterologous nucleic acid is a heterologous nucleicacid coding for a peptide, oligopeptide, polypeptide or protein.
 20. Thebeta-herpesvirus according to claim 19, wherein the peptide,oligopeptide, polypeptide or protein constitutes or comprises at leastone antigen, wherein the antigen is an antigen selected from the groupconsisting of tumor antigens, tumor associated antigens, viral antigens,bacterial antigens and parasite antigens.
 21. The beta-herpesvirusaccording to claim 20, wherein the viral antigen is an antigen derivedfrom a virus, wherein the virus is selected from the group consisting ofHIV, Influenza, HPV and RSV.
 22. The beta-herpesvirus according to claim20, wherein the bacterial antigen is an antigen derived from abacterium, wherein the bacterium is selected from the group consistingof mycobacterium, Helicobacter pylori and Listeria.
 23. Thebeta-herpesvirus according to claim 20, wherein the parasite antigen isan antigen derived from a parasite, wherein the parasite is selectedfrom the group consisting of Plasmodium.
 24. A method for the treatmentor prevention of a disease comprising administering to a subject thebeta-herpesvirus according to claim
 1. 25. The method according to claim24, wherein the disease is a disease or condition which is associatedwith beta-herpesvirus infection.
 26. The method according to claim 24,wherein the disease is a disease selected from the group consisting ofbacterial disease, viral disease, parasite disease and tumors, whereinthe beta-herpesvirus is expressing a bacterial antigen, a viral antigen,a parasite antigen or a tumor antigen.
 27. A method for the vaccinationof a subject against a diseases, comprising the administration to thesubject of the beta-herpesvirus according to claim
 1. 28. A nucleic acidcoding for a beta-herpesvirus as defined in claim
 1. 29. An expressionvector, comprising the nucleic acid according to claim
 28. 30. Apharmaceutical composition comprising a beta-herpesvirus according toclaim 1 and/or a nucleic acid according to claim 28, and apharmaceutically acceptable carrier.